1H-PYRAZOLO[4,3-d]PYRIMIDINE COMPOUNDS AS TOLL-LIKE RECEPTOR 7 (TLR7) AGONISTS AND METHODS AND USES THEREFOR

ABSTRACT

Such compounds can be used in cancer treatment, especially in combination with an anti-cancer immunotherapy agent, or as a vaccine adjuvant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 62/714,238, filed Aug. 3, 2018; thedisclosure of which is incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

This disclosure relates to Toll-like receptor 7 (“TLR7”) agonists andconjugates thereof, and methods for the preparation and use of suchagonists and their conjugates.

Toll-like receptors (“TLRs”) are receptors that recognizepathogen-associated molecular patterns (“PAMPs”), which are smallmolecular motifs conserved in certain classes of pathogens. TLRs can belocated either on a cell's surface or intracellularly. Activation of aTLR by the binding of its cognate PAMP signals the presence of theassociated pathogen inside the host—i.e., an infection—and stimulatesthe host's immune system to fight the infection. Humans have 10 TLRs,named TLR1, TLR2, TLR3, and so on.

The activation of a TLR—with TLR7 being the most studied—by an agonistcan have a positive effect on the action of vaccines and immunotherapyagents in treating a variety of conditions other than actual pathogeninfection, by stimulating the immune response overall. Thus, there isconsiderable interest in the use of TLR7 agonists as vaccine adjuvantsor as enhancers in cancer immunotherapy. See, for example, Vasilakos andTomai 2013, Sato-Kaneko et al. 2017, Smits et al. 2008, and Ota et al.2019.

TLR7, an intracellular receptor located on the membrane of endosomes,recognizes PAMPs associated with single-stranded RNA viruses. Itsactivation induces secretion of Type I interferons such as IFNα and IFNβ(Lund et al. 2004). TLR7 has two binding sites, one for single strandedRNA ligands (Berghöfer et al. 2007) and one for small molecules such asguanosine (Zhang et al. 2016).

TLR7 can bind to, and be activated by, guanosine-like synthetic agonistssuch as imiquimod, resiquimod, and gardiquimod, which are based on a1H-imidazo[4,5-c]quinoline scaffold. For a review of small-molecule TLR7agonists, see Cortez and Va 2018.

Synthetic TLR7 agonists based on a pteridinone molecular scaffold arealso known, as exemplified by vesatolimod (Desai et al. 2015).

Other synthetic TLR7 agonists based on a purine-like scaffold have beendisclosed, frequently according to the general formula (A):

where R, R′, and R″ are structural variables, with R″ typicallycontaining an unsubstituted or substituted aromatic or heteroaromaticring.

Disclosures of bioactive molecules having a purine-like scaffold andtheir uses in treating conditions such as fibrosis, inflammatorydisorders, cancer, or pathogenic infections include: Akinbobuyi et al.2015 and 2016; Barberis et al. 2012; Carson et al. 2014; Ding et al.2016, 2017a, and 2017b; Graupe et al. 2015; Hashimoto et al. 2009; He etal. 2019a and 2019b; Holldack et al. 2012; Isobe et al. 2009a and 2012;Poudel et al. 2019a and 2019b; Pryde 2010; and Young et al. 2019.

The group R″ can be pyridyl: Bonfanti et al. 2015a and 2015b; Halcomb etal. 2015; Hirota et al. 2000; Isobe et al. 2002, 2004, 2006, 2009a,2009b, 2011, and 2012; Kasibhatla et al. 2007; Koga-Yamakawa et al.2013; Musmuca et al. 2009; Nakamura 2012; Ogita et al. 2007; and Yu etal. 2013.

There are disclosures of related molecules in which the 6,5-fused ringsystem of formula (A)—a pyrimidine six member ring fused to an imidazolefive member ring—is modified. (a) Dellaria et al. 2007, Jones et al.2010 and 2012, and Pilatte et al. 2017 disclose compounds in which thepyrimidine ring is replaced by a pyridine ring. (b) Chen et al. 2011,Coe et al. 2017, and Zhang et al. 2018 disclose compounds in which theimidazole ring is replaced by a pyrazole ring. (c) Cortez et al. 2017and 2018; Li et al. 2018; and McGowan et al. 2016a, 2016b, and 2017disclose compounds in which the imidazole ring is replaced by a pyrrolering.

Bonfanti et al. 2015b and 2016 and Purandare et al. 2019 disclose TLR7modulators in which the two rings of a purine moiety are spanned by amacrocycle:

A TLR7 agonist can be conjugated to a partner molecule, which can be,for example, a phospholipid, a poly(ethylene glycol) (“PEG”), anantibody, or another TLR (commonly TLR2). Exemplary disclosures include:Carson et al. 2013, 2015, and 2016, Chan et al. 2009 and 2011, Cortez etal. 2017, Gadd et al. 2015, Lioux et al. 2016, Maj et al. 2015, Vemejoulet al. 2014, and Zurawski et al. 2012. A frequent conjugation site is atthe R″ group of formula (A).

Jensen et al. 2015 discloses the use of cationic lipid vehicles for thedelivery of TLR7 agonists.

Some TLR7 agonists, including resiquimod are dual TLR7/TLR8 agonists.See, for example, Beesu et al. 2017, Embrechts et al. 2018, Lioux et al.2016, and Vemejoul et al. 2014.

Full citations for the documents cited herein by first author orinventor and year are listed at the end of this specification.

BRIEF SUMMARY OF THE DISCLOSURE

This specification relates to compounds having a1H-pyrazolo[4,3d]pyrimidine aromatic system, having activity as TLR7agonists.

In one aspect, there is provided a compound with a structure accordingto formula I

wherein

-   each X¹ is independently N or CR²;-   X² is O, CH₂, NH, S, or N(C₁-C₃ alkyl);-   R¹ is H, CH₃(CH₂)₁₋₃, CH₃(CH₂)₀₋₁O(CH₂)₂₋₃, CH₃(CH₂)₀₋₃C(═O),    CH₃(CH₂)₀₋₁O(CH₂)₂₋₃C(═O),

-   R² is H, O(C₁-C₃ alkyl), C₁-C₃ alkyl, Cl, F, or CN;-   R³ is H, halo, OH, CN, NH₂, NH(C₁-C₅ alkyl), N(C₁-C₅ alkyl)₂,    NH(CH₂)₀₋₁(C₃-C₆ cycloalkyl), NH(C₄-C₈ bicycloalkyl), NH(C₆-C₁₀    spirocycloalkyl), N(C₃-C₆ cycloalkyl)₂, NH(CH₂)₁₋₃(aryl),    N((CH₂)₁₋₃(aryl))₂, a cyclic amine moiety having the structure

a 6-membered aromatic or heteroaromatic moiety or a 5-memberedheteroaromatic moiety;

wherein

-   -   an alkyl, cycloalkyl, bicycloalkyl, spirocycloalkyl, cyclic        amine, 6-membered aromatic or heteroaromatic, or 5-membered        heteroaromatic moiety is optionally substituted with one or more        substituents selected from OH, halo, CN, (C₁-C₃ alkyl), O(C₁-C₃        alkyl), C(═O)(Me), SO₂(C₁-C₃ alkyl), C(═O)(Et), NH₂, NH(Me),        N(Me)₂, NH(Et), N(Et)₂, and N(C₁-C₃ alkyl), (CH₂)₁₋₂OH,        (CH₂)₁₋₂OMe; and    -   a cycloalkyl, bicycloalkyl, spirocycloalkyl, or cyclic amine        moiety may have a CH₂ group replaced by O, S, NH, N(C₁-C₃        alkyl), or N(Boc);

-   m is 0 or 1;

-   and

-   n is 1, 2, or 3;

-   or a pharmaceutically acceptable salt thereof.

Compounds disclosed herein have activity as TLR7 agonists and some canbe conjugated to an antibody for targeted delivery to a target tissue ororgan of intended action. They can also be PEGylated, to modulate theirpharmaceutical properties.

Compounds disclosed herein, or their conjugates or their PEGylatedderivatives, can be used in the treatment of a subject suffering from acondition amenable to treatment by activation of the immune system, byadministering to such subject a therapeutically effective amount of sucha compound or a conjugate thereof or a PEGylated derivative thereof,especially in combination with a vaccine or a cancer immunotherapyagent.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIGS. 1, 2A, 2B, 3A, 3B, 4A, 4B, 5, 6A, 6B, 7, 8, 9, 10, 11, 12, 13 and14 show reaction schemes for preparing compounds disclosed herein.

FIGS. 15 and 16 show schemes for the attachment of linkers to compoundsof this disclosure, rendering them suitable for conjugation.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Antibody” means whole antibodies and any antigen binding fragment(i.e., “antigen-binding portion”) or single chain variants thereof. Awhole, or full length, antibody is a protein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain comprises a heavy chain variable region (V_(H))and a heavy chain constant region comprising three domains, CH₁, CH₂ andCH₃. Each light chain comprises a light chain variable region (V_(L) orV_(k)) and a light chain constant region comprising one single domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with more conserved framework regions (FRs). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged from amino-to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions contain a binding domain thatinteracts with an antigen. The constant regions may mediate the bindingof the antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. An antibody is said to “specificallybind” to an antigen X if the antibody binds to antigen X with a K_(D) of5×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, more preferably6×10⁻⁹ M or less, more preferably 3×10⁻⁹ M or less, even more preferably2×10⁻⁹ M or less. The antibody can be chimeric, humanized, or,preferably, human. The heavy chain constant region can be engineered toaffect glycosylation type or extent, to extend antibody half-life, toenhance or reduce interactions with effector cells or the complementsystem, or to modulate some other property. The engineering can beaccomplished by replacement, addition, or deletion of one or more aminoacids or by replacement of a domain with a domain from anotherimmunoglobulin type, or a combination of the foregoing.

“Antigen binding fragment” and “antigen binding portion” of an antibody(or simply “antibody portion” or “antibody fragment”) mean one or morefragments of an antibody that retain the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody, suchas (i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and CH₁ domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fab′ fragment, which is essentially an Fabwith part of the hinge region (see, for example, Abbas et al., Cellularand Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) a Fdfragment consisting of the V_(H) and CH₁ domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Preferred antigen binding fragments are Fab, F(ab′)₂, Fab′, Fv, and Fdfragments. Furthermore, although the two domains of the Fv fragment,V_(L) and V_(H), are encoded by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv, or scFv); see,e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also encompassed within the term “antigen-binding portion” of anantibody.

Unless indicated otherwise—for example by reference to the linearnumbering in a SEQ ID NO: listing—references to the numbering of aminoacid positions in an antibody heavy or light chain variable region(V_(H) or V_(L)) are according to the Kabat system (Kabat et al.,“Sequences of proteins of immunological interest, 5th ed., Pub. No.91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991,hereinafter “Kabat”) and references to the numbering of amino acidpositions in an antibody heavy or light chain constant region (C_(H1),C_(H2), C_(H3), or C_(L)) are according to the EU index as set forth inKabat. See Lazar et al., US 2008/0248028 A1, the disclosure of which isincorporated herein by reference, for examples of such usage. Further,the ImMunoGeneTics Information System (IMGT) provides at its website atable entitled “IMGT Scientific Chart: Correspondence between CNumberings” showing the correspondence between its numbering system, EUnumbering, and Kabat numbering for the heavy chain constant region.

An “isolated antibody” means an antibody that is substantially free ofother antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds antigen X is substantiallyfree of antibodies that specifically bind antigens other than antigenX). An isolated antibody that specifically binds antigen X may, however,have cross-reactivity to other antigens, such as antigen X moleculesfrom other species. In certain embodiments, an isolated antibodyspecifically binds to human antigen X and does not cross-react withother (non-human) antigen X antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

“Monoclonal antibody” or “monoclonal antibody composition” means apreparation of antibody molecules of single molecular composition, whichdisplays a single binding specificity and affinity for a particularepitope.

“Human antibody” means an antibody having variable regions in which boththe framework and CDR regions (and the constant region, if present) arederived from human germ-line immunoglobulin sequences. Human antibodiesmay include later modifications, including natural or syntheticmodifications. Human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

“Human monoclonal antibody” means an antibody displaying a singlebinding specificity, which has variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, human monoclonal antibodies are producedby a hybridoma that includes a B cell obtained from a transgenicnonhuman animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a light chain transgene fused to animmortalized cell.

“Aliphatic” means a straight- or branched-chain, saturated orunsaturated, non-aromatic hydrocarbon moiety having the specified numberof carbon atoms (e.g., as in “C₃ aliphatic,” “C₁₋₅ aliphatic,” “C₁-C₅aliphatic,” or “C₁ to C₅ aliphatic,” the latter three phrases beingsynonymous for an aliphatic moiety having from 1 to 5 carbon atoms) or,where the number of carbon atoms is not explicitly specified, from 1 to4 carbon atoms (2 to 4 carbons in the instance of unsaturated aliphaticmoieties). A similar understanding is applied to the number of carbonsin other types, as in C₂₋₄ alkene, C₄-C₇ cycloaliphatic, etc. In asimilar vein, a term such as “(CH₂)₁₋₃” is to be understand as shorthandfor the subscript being 1, 2, or 3, so that such term represents CH₂,CH₂CH₂, and CH₂CH₂CH₂.

“Alkyl” means a saturated aliphatic moiety, with the same convention fordesignating the number of carbon atoms being applicable. By way ofillustration, C₁-C₄ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl,and the like. “Alkylene” means a divalent counterpart of an alkyl group,such as CH₂CH₂, CH₂CH₂CH₂, and CH₂CH₂CH₂CH₂.

“Alkenyl” means an aliphatic moiety having at least one carbon-carbondouble bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkenylmoieties include, but are not limited to, ethenyl (vinyl), 2-propenyl(allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E- (or Z-)2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl) and the like.

“Alkynyl” means an aliphatic moiety having at least one carbon-carbontriple bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkynylgroups include ethynyl (acetylenyl), propargyl (prop-2-ynyl),1-propynyl, but-2-ynyl, and the like.

“Cycloaliphatic” means a saturated or unsaturated, non-aromatichydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to8 (preferably from 3 to 6) carbon atoms. “Cycloalkyl” means acycloaliphatic moiety in which each ring is saturated. “Cycloalkenyl”means a cycloaliphatic moiety in which at least one ring has at leastone carbon-carbon double bond. “Cycloalkynyl” means a cycloaliphaticmoiety in which at least one ring has at least one carbon-carbon triplebond. By way of illustration, cycloaliphatic moieties include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl.Preferred cycloaliphatic moieties are cycloalkyl ones, especiallycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. “Cycloalkylene”means a divalent counterpart of a cycloalkyl group. Similarly,“bicycloalkylene” and “spirocycloalkylene” (or “spiroalkylene”) refer todivalent counterparts of a bicycloalkyl and spirocycloalkyl/spiroalkylgroup.

“Heterocycloaliphatic” means a cycloaliphatic moiety wherein, in atleast one ring thereof, up to three (preferably 1 to 2) carbons havebeen replaced with a heteroatom independently selected from N, O, or S,where the N and S optionally may be oxidized and the N optionally may bequaternized. Preferred cycloaliphatic moieties consist of one ring, 5-to 6-membered in size. Similarly, “heterocycloalkyl,”“heterocycloalkenyl,” and “heterocycloalkynyl” means a cycloalkyl,cycloalkenyl, or cycloalkynyl moiety, respectively, in which at leastone ring thereof has been so modified. Exemplary heterocycloaliphaticmoieties include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl,tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone,morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinylsulfone, 1,3-dioxolanyl, tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl,thietanyl, and the like. “Heterocycloalkylene” means a divalentcounterpart of a heterocycloalkyl group.

“Alkoxy,” “aryloxy,” “alkylthio,” and “arylthio” mean —O(alkyl),—O(aryl), —S(alkyl), and —S(aryl), respectively. Examples are methoxy,phenoxy, methylthio, and phenylthio, respectively.

“Halogen” or “halo” means fluorine, chlorine, bromine or iodine, unlessa narrower meaning is indicated.

“Aryl” means a hydrocarbon moiety having a mono-, bi-, or tricyclic ringsystem (preferably monocyclic) wherein each ring has from 3 to 7 carbonatoms and at least one ring is aromatic. The rings in the ring systemmay be fused to each other (as in naphthyl) or bonded to each other (asin biphenyl) and may be fused or bonded to non-aromatic rings (as inindanyl or cyclohexylphenyl). By way of further illustration, arylmoieties include, but are not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracenyl, andacenaphthyl. “Arylene” means a divalent counterpart of an aryl group,for example 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

“Heteroaryl” means a moiety having a mono-, bi-, or tricyclic ringsystem (preferably 5- to 7-membered monocyclic) wherein each ring hasfrom 3 to 7 carbon atoms and at least one ring is an aromatic ringcontaining from 1 to 4 heteroatoms independently selected from N, O, orS, where the N and S optionally may be oxidized and the N optionally maybe quaternized. Such at least one heteroatom containing aromatic ringmay be fused to other types of rings (as in benzofuranyl ortetrahydroisoquinolyl) or directly bonded to other types of rings (as inphenylpy-ridyl or 2-cyclopentylpyridyl). By way of further illustration,heteroaryl moieties include pyrrolyl, furanyl, thiophenyl (thienyl),imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl,triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, quinolinyl, isoquinolynyl, quinazolinyl, cinnolinyl,quinozalinyl, naphthyridinyl, benzofuranyl, indolyl, benzothiophenyl,oxadiazolyl, thiadiazolyl, phenothiazolyl, benzimidazolyl,benzotriazolyl, dibenzofuranyl, carbazolyl, dibenzothiophenyl,acridinyl, and the like. “Heteroarylene” means a divalent counterpart ofa heteroaryl group.

Where it is indicated that a moiety may be substituted, such as by useof “unsubstituted or substituted” or “optionally substituted” phrasingas in “unsubstituted or substituted C₁-C₅ alkyl” or “optionallysubstituted heteroaryl,” such moiety may have one or more independentlyselected substituents, preferably one to five in number, more preferablyone or two in number. Substituents and substitution patterns can beselected by one of ordinary skill in the art, having regard for themoiety to which the substituent is attached, to provide compounds thatare chemically stable and that can be synthesized by techniques known inthe art as well as the methods set forth herein. Where a moiety isidentified as being “unsubstituted or substituted” or “optionallysubstituted,” in a preferred embodiment such moiety is unsubstituted.

“Arylalkyl,” (heterocycloaliphatic)alkyl,” “arylalkenyl,” “arylalkynyl,”“biarylalkyl,” and the like mean an alkyl, alkenyl, or alkynyl moiety,as the case may be, substituted with an aryl, heterocycloaliphatic,biaryl, etc., moiety, as the case may be, with the open (unsatisfied)valence at the alkyl, alkenyl, or alkynyl moiety, for example as inbenzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.Conversely, “alkylaryl,” “alkenylcycloalkyl,” and the like mean an aryl,cycloalkyl, etc., moiety, as the case may be, substituted with an alkyl,alkenyl, etc., moiety, as the case may be, for example as inmethylphenyl (tolyl) or allylcyclohexyl. “Hydroxyalkyl,” “haloalkyl,”“alkylaryl,” “cyanoaryl,” and the like mean an alkyl, aryl, etc.,moiety, as the case may be, substituted with one or more of theidentified substituent (hydroxyl, halo, etc., as the case may be).

For example, permissible substituents include, but are not limited to,alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl,aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especiallyfluoro), haloalkyl (especially trifluoromethyl), hydroxyl, hydroxyalkyl(especially hydroxyethyl), cyano, nitro, alkoxy, —O(hydroxyalkyl),—O(haloalkyl) (especially —OCF₃), —O(cycloalkyl), —O(heterocycloalkyl),—O(aryl), alkylthio, arylthio, ═O, ═NH, ═N(alkyl), ═NOH, ═NO(alkyl),—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl),—SO₂N(alkyl)₂, and the like.

Where the moiety being substituted is an aliphatic moiety, preferredsubstituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic,halo, hydroxyl, cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl),—O(cycloalkyl), —O(heterocycloalkyl), —O(aryl), alkylthio, arylthio, ═O,═NH, ═N(alkyl), ═NOH, ═NO(alkyl), —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(═O)alkyl, —S(cycloalkyl), —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are halo, hydroxyl, cyano,nitro, alkoxy, —O(aryl), ═O, ═NOH, ═NO(alkyl), —OC(═O)(alkyl),—OC(═O)O(alkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂, azido,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are phenyl, cyano, halo, hydroxyl, nitro,C₁-C₄alkyoxy, O(C₂-C₄ alkylene)OH, and O(C₂-C₄ alkylene)halo.

Where the moiety being substituted is a cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituentsare alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl,cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl), —O(aryl),—O(cycloalkyl), —O(heterocycloalkyl), alkylthio, arylthio,—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are alkyl, alkenyl, halo,haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy,—O(hydroxyalkyl), —C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH,—C(═O)O(alkyl), —C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl),—C(═O)N(alkyl)₂, —OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are C₁-C₄ alkyl, cyano, nitro, halo, andC₁-C₄alkoxy.

Where a range is stated, as in “C₁-C₅ alkyl” or “5 to 10%,” such rangeincludes the end points of the range, as in C₁ and C₅ in the firstinstance and 5% and 10% in the second instance.

Unless particular stereoisomers are specifically indicated (e.g., by abolded or dashed bond at a relevant stereocenter in a structuralformula, by depiction of a double bond as having E or Z configuration ina structural formula, or by use stereochemistry-designating nomenclatureor symbols), all stereoisomers are included within the scope of theinvention, as pure compounds as well as mixtures thereof. Unlessotherwise indicated, racemates, individual enantiomers (whetheroptically pure or partially resolved), diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythis invention.

Those skilled in the art will appreciate that compounds may havetautomeric forms (e.g., keto and enol forms), resonance forms, andzwitterionic forms that are equivalent to those depicted in thestructural formulae used herein and that the structural formulaeencompass such tautomeric, resonance, or zwitterionic forms.

“Pharmaceutically acceptable ester” means an ester that hydrolyzes invivo (for example in the human body) to produce the parent compound or asalt thereof or has per se activity similar to that of the parentcompound. Suitable esters include C₁-C₅ alkyl, C₂-C₅ alkenyl or C₂-C₅alkynyl esters, especially methyl, ethyl or n-propyl.

“Pharmaceutically acceptable salt” means a salt of a compound suitablefor pharmaceutical formulation. Where a compound has one or more basicgroups, the salt can be an acid addition salt, such as a sulfate,hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate,pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate,methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate,suberate, tosylate, and the like. Where a compound has one or moreacidic groups, the salt can be a salt such as a calcium salt, potassiumsalt, magnesium salt, meglumine salt, ammonium salt, zinc salt,piperazine salt, tromethamine salt, lithium salt, choline salt,diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodiumsalt, tetramethylammonium salt, and the like. Polymorphic crystallineforms and solvates are also encompassed within the scope of thisinvention.

“Subject” refers to an animal, including, but not limited to, a primate(e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit,rat, or mouse. The terms “subject” and “patient” are usedinterchangeably herein in reference, for example, to a mammaliansubject, such as a human.

The terms “treat,” “treating,” and “treatment,” in the context oftreating a disease or disorder, are meant to include alleviating orabrogating a disorder, disease, or condition, or one or more of thesymptoms associated with the disorder, disease, or condition; or toslowing the progression, spread or worsening of a disease, disorder orcondition or of one or more symptoms thereof. The “treatment of cancer”,refers to one or more of the following effects: (1) inhibition, to someextent, of tumor growth, including, (i) slowing down and (ii) completegrowth arrest; (2) reduction in the number of tumor cells; (3)maintaining tumor size; (4) reduction in tumor size; (5) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of tumor cell infiltration into peripheral organs; (6) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of metastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion and/or (8) relief, to some extent, of the severity or number ofone or more symptoms associated with the disorder.

In the formulae of this specification, a wavy line (

) transverse to a bond or an asterisk (*) at the end of the bond denotesa covalent attachment site. For instance, a statement that R is

or that R is

in the formula

means

In the formulae of this specification, a bond traversing an aromaticring between two carbons thereof means that the group attached to thebond may be located at any of the positions of the aromatic ring madeavailable by removal of the hydrogen that is implicitly there. By way ofillustration, the formula

represents

In other illustrations,

represents

represents

Those skilled in the art will appreciate that certain structures can bedrawn in one tautomeric form or another—for example, keto versusenol—and that the two forms are equivalent.

Compounds

In one embodiment, either each X¹ is CR² or not more than two X¹'s are Nin the moiety

Examples of such are

A preferred example is

equals

Suitable groups R¹ include:

Preferably the group R¹ is

Examples of suitable groups R³ include Cl, OH,

Examples of where the group R³ has the structure

(including instances with one or more methylene (CH₂) groups optionallyreplaced by one or more of O, S, SO₂, NH, C(═O), N(C₁-C₃ alkyl),NC(═O)(C₁-C₃ alkyl), or N(Boc), or has another ring fused thereto, asdisclosed hereinabove) are:

In another embodiment, R³ is selected from the group consisting of

In one embodiment of formula I, m is 0, in which case the formulasimplifies to formula I′:

Another embodiment of compounds according to formula I is represented byformula Ia:

where R¹ and R³ are as defined in respect of formulae I hereinabove.

An embodiment of compounds according to formula I wherein m is 1 isrepresented by formula Ib, wherein R¹, R³ and X¹ are as defined inrespect of formula I hereinabove.

Preferably, in formula Ib

or, more preferably

Specific examples of compounds per in this specification are listedfollowing. Methods for their preparation and their properties areprovided in the experimental sections hereinbelow.

No. Structure 800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

817

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820

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825

826

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828

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840

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849

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871

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875

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878

879

Conjugates General

TLR7 agonists disclosed herein can be delivered to the site of intendedaction by localized administration or by targeted delivery in aconjugate with a targeting moiety. Preferably, the targeting moiety isan antibody or antigen binding portion thereof and its antigen is foundat the locality of intended action, for example a tumor associatedantigen if the intended site of action is at a tumor (cancer).Preferably, the tumor associated antigen is uniquely expressed oroverexpressed by the cancer cell, compared to a normal cell. The tumorassociated antigen can be located on the surface of the cancer cell orsecreted by the cancer cell into its environs.

In one aspect, there is provided a conjugate comprising compound of thisinvention and a ligand, represented by formula (IV)

[D(X^(D))_(a)(C)_(c)(X^(Z))_(b)]_(m)Z  (IV)

where Z is a targeting moiety, D is an agonist of this invention, and—(X^(D))_(a)C(X^(Z))_(b)— are collectively referred to as a “linkermoiety” or “linker” because they link Z and D. Within the linker, C is acleavable group designed to be cleaved at or near the site of intendedbiological action of D; X^(D) and X^(Z) are spacer moieties (or“spacers”) that space apart D and C and C and Z, respectively;subscripts a, b, and c are independently 0 or 1 (that is, the presenceof X^(D), X^(Z) and C are optional). Subscript m is 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 (preferably 1, 2, 3, or 4). D, X^(D), C, X^(Z) and Z aremore fully described hereinbelow.

By binding to a target tissue or cell where its antigen or receptor islocated, Z directs the conjugate there. Cleavage of group C at thetarget tissue or cell releases D to exert its effect locally. In thismanner, precise delivery of D is achieved at the site of intendedaction, reducing the dosage needed. Also, D is normally biologicallyinactive (or significantly less active) in its conjugated state, therebyreducing off-target effects.

As reflected by the subscript m, each Z can conjugate with more than oneD, depending on the number of sites Z has available for conjugation andthe experimental conditions employed. Those skilled in the art willappreciate that, while each individual Z is conjugated to an integernumber of Ds, a preparation of the conjugate may analyze for anon-integer ratio of D to Z, reflecting a statistical average. Thisratio is referred to as the substitution ratio (“SR”) or thedrug-antibody ratio (“DAR”).

Targeting Moiety Z

Preferably, targeting moiety Z is an antibody. For convenience andbrevity and not by way of limitation, the detailed discussion in thisspecification about Z and its conjugates is written in the context ofits being an antibody, but those skilled in the art will understand thatother types of Z can be conjugated, mutatis mutandis. For example,conjugates with folic acid as the targeting moiety can target cellshaving the folate receptor on their surfaces (Leamon et al., Cancer Res.2008, 68 (23), 9839). For the same reasons, the detailed discussion inthis specification is primarily written in terms of a 1:1 ratio of Z toD (m=1).

Antibodies that can be used in conjugates of this invention includethose recognizing the following antigens: mesothelin, prostate specificmembrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H3, B7H4 (also knownas 08E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, fucosyl-GM1,CTLA-4, and CD44. The antibody can be animal (e.g., murine), chimeric,humanized, or, preferably, human. The antibody preferably is monoclonal,especially a monoclonal human antibody. The preparation of humanmonoclonal antibodies against some of the aforementioned antigens isdisclosed in Korman et al., U.S. Pat. No. 8,609,816 B2 (2013; B7H4, alsoknown as 08E; in particular antibodies 2A7, 1G11, and 2F9); Rao-Naik etal., U.S. Pat. No. 8,097,703 B2 (2012; CD19; in particular antibodies5G7, 13F1, 46E8, 21D4, 21D4a, 47G4, 27F3, and 3C10); King et al., U.S.Pat. No. 8,481,683 B2 (2013; CD22; in particular antibodies 12C5, 19A3,16F7, and 23C6); Keler et al., U.S. Pat. No. 7,387,776 B2 (2008; CD30;in particular antibodies 5F11, 2H9, and 17G1); Terrett et al., U.S. Pat.No. 8,124,738 B2 (2012; CD70; in particular antibodies 2H5, 10B4, 8B5,18E7, and 69A7); Korman et al., U.S. Pat. No. 6,984,720 B1 (2006;CTLA-4; in particular antibodies 10D1, 4B6, and 1E2); Korman et al.,U.S. Pat. No. 8,008,449 B2 (2011; PD-1; in particular antibodies 17D8,2D3, 4H1, 5C4, 4A11, 7D3, and 5F4); Huang et al., US 2009/0297438 A1(2009; PSMA. in particular antibodies 1C3, 2A10, 2F5, 2C6); Cardarelliet al., U.S. Pat. No. 7,875,278 B2 (2011; PSMA; in particular antibodies4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5, and 1C3); Terrett et al.,U.S. Pat. No. 8,222,375 B2 (2012; PTK7; in particular antibodies 3G8,4D5, 12C6, 12C6a, and 7C8); Harkins et al., U.S. Pat. No. 7,335,748 B2(2008; RG1; in particular antibodies A, B, C, and D); Terrett et al.,U.S. Pat. No. 8,268,970 B2 (2012; mesothelin; in particular antibodies3C10, 6A4, and 7B1); Xu et al., US 2010/0092484 A1 (2010; CD44; inparticular antibodies 14G9.B8.B4, 2D1.A3.D12, and 1A9.A6.B9); Deshpandeet al., U.S. Pat. No. 8,258,266 B2 (2012; IP10; in particular antibodies1D4, 1E1, 2G1, 3C4, 6A5, 6A8, 7C10, 8F6, 10A12, 10A12S, and 13C4); Kuhneet al., U.S. Pat. No. 8,450,464 B2 (2013; CXCR4; in particularantibodies F7, F9, D1, and E2); and Korman et al., U.S. Pat. No.7,943,743 B2 (2011; PD-L1; in particular antibodies 3G10, 12A4, 10A5,5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4); the disclosures of whichare incorporated herein by reference. Preferably, the antibody is ananti-mesothelin antibody.

In addition to being an antibody, Z can also be an antibody fragment(such as Fab, Fab′, F(ab′)₂, Fd, or Fv) or antibody mimetic, such as anaffibody, a domain antibody (dAb), a nanobody, a unibody, a DARPin, ananticalin, a versabody, a duocalin, a lipocalin, or an avimer.

Any one of several different reactive groups on Z can be a conjugationsite, including ε-amino groups in lysine residues, pendant carbohydratemoieties, carboxylic acid groups on aspartic or glutamic acid sidechains, cysteine-cysteine disulfide groups, and cysteine thiol groups.For reviews on antibody reactive groups suitable for conjugation, see,e.g., Garnett, Adv. Drug Delivery Rev. 2001, 53, 171-216 and Dubowchikand Walker, Pharmacology & Therapeutics 1999, 83, 67-123, thedisclosures of which are incorporated herein by reference.

Most antibodies have multiple lysine residues, which can be conjugatedvia their ε-amino groups via amide, urea, thiourea, or carbamate bonds.

A thiol (—SH) group in the side chain of a cysteine can be used to forma conjugate by several methods. It can be used to form a disulfide bondbetween it and a thiol group on the linker. Another method is via itsMichael addition to a maleimide group on the linker.

Typically, although antibodies have cysteine residues, they lack freethiol groups because all their cysteines are engaged in intra- orinter-chain disulfide bonds. To generate a free thiol group, a nativedisulfide group can be reduced. See, e.g., Packard et al., Biochemistry1986, 25, 3548; King et al., Cancer Res. 1994, 54, 6176; and Doronina etal., Nature Biotechnol. 2003, 21, 778. Alternatively, a cysteine havinga free —SH group can be introduced by mutating the antibody,substituting a cysteine for another amino acid or inserting one into thepolypeptide chain. See, for example, Eigenbrot et al., U.S. Pat. No.7,521,541 B2 (2009); Chilkoti et al., Bioconjugate Chem. 1994, 5, 504;Umovitz et al., U.S. Pat. No. 4,698,420 (1987); Stimmel et al., J. Biol.Chem. 2000, 275, 30445; Bam et al., U.S. Pat. No. 7,311,902 B2 (2007);Kuan et al., J. Biol. Chem. 1994, 269, 7610; Poon et al., J. Biol. Chem.1995, 270, 8571; Junutula et al., Nature Biotechnology 2008, 26, 925 andRajpal et al., U.S. Provisional Application No. 62/270,245, filed Dec.21, 2015. In yet another approach, a cysteine is added to the C-terminusof the heavy of light chain. See, e.g., Liu et al., U.S. Pat. No.8,865,875 B2 (2014); Cumber et al., J. Immunol. 1992, 149, 120; King etal, Cancer Res. 1994, 54, 6176; Li et al., Bioconjugate Chem. 2002, 13,985; Yang et al., Protein Engineering 2003, 16, 761; and Olafson et al.,Protein Engineering Design & Selection 2004, 17, 21. The disclosures ofthe documents cited in this paragraph are incorporated herein byreference.

Linkers and their Components

As noted above, the linker comprises up to three elements: a cleavablegroup C and optional spacers X^(Z) and X^(D).

Group C is cleavable under physiological conditions. Preferably it isrelatively stable while the conjugate is in circulation in the blood,but is readily cleaved once the conjugate reaches its site of intendedaction.

A preferred group C is a peptide that is cleaved selectively by aprotease inside the target cell, as opposed to by a protease in theserum. Typically, the peptide comprises from 1 to 20 amino acids,preferably from 1 to 6 amino acids, more preferably from 2 to 3 aminoacids. The amino acid(s) can be natural and/or non-natural α-aminoacids. Natural amino acids are those encoded by the genetic code, aswell as amino acids derived therefrom, e.g., hydroxyproline,γ-carboxyglutamate, citrulline, and O-phosphoserine. In thisspecification, the term “amino acid” also includes amino acid analogsand mimetics. Analogs are compounds having the same general H₂N(R)CHCO₂Hstructure of a natural amino acid, except that the R group is not onefound among the natural amino acids. Examples of analogs includehomoserine, norleucine, methionine-sulfoxide, and methionine methylsulfonium. An amino acid mimetic is a compound that has a structuredifferent from the general chemical structure of an α-amino acid butfunctions in a manner similar to one. The amino acid can be of the “L”stereochemistry of the genetically encoded amino acids, as well as ofthe enantiomeric “D” stereochemistry.

Preferably, C contains an amino acid sequence that is a cleavagerecognition sequence for a protease. Many cleavage recognition sequencesare known in the art. See, e.g., Matayoshi et al. Science 247: 954(1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth.Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994);Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol.244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); thedisclosures of which are incorporated herein by reference.

A group C can be chosen such that it is cleaved by a protease present inthe extracellular matrix in the vicinity of a cancer, e.g., a proteasereleased by nearby dying cancer cells or a tumor-associated proteasesecreted by cancer cells. Exemplary extracellular tumor-associatedproteases are plasmin, matrix metalloproteases (MMP), thimetoligopeptidase (TOP) and CD10. See, e.g., Trouet et al., U.S. Pat. No.7,402,556 B2 (2008); Dubois et al., U.S. Pat. No. 7,425,541 B2 (2008);and Bebbington et al., U.S. Pat. No. 6,897,034 B2 (2005). Cathepsin D,normally lysosomal enzyme found inside cells, is sometimes found in theenvirons of a tumor, possibly released by dying cancer cells.

For conjugates designed to be by an enzyme, C preferably comprises anamino acid sequence selected for cleavage by proteases such cathepsinsB, C, D, H, L and S, especially cathepsin B. Exemplary cathepsin Bcleavable peptides include Val-Ala, Val-Cit, Val-Lys, Lys-Val-Ala,Asp-Val-Ala, Val-Ala, Lys-Val-Cit, Ala-Val-Cit, Val-Gly, Val-Gln, andAsp-Val-Cit. (Herein, amino acid sequences are written in the N-to-Cdirection, as in H2N-AA²-AA¹-CO₂H, unless the context clearly indicatesotherwise.) See Dubowchik et al., Biorg. Med. Chem. Lett. 1998, 8, 3341;Dubowchik et al., Bioorg. Med. Chem. Lett. 1998, 8, 3347; and Dubowchiket al., Bioconjugate Chem. 2002, 13, 855; the disclosures of which areincorporated by reference.

Another enzyme that can be utilized for cleaving peptidyl linkers islegumain, a lysosomal cysteine protease that preferentially cleaves atAla-Ala-Asn.

In one embodiment, Group C is a peptide comprising a two-amino acidsequence -AA²-AA¹- wherein AA¹ is lysine, arginine, or citrulline andAA² is phenylalanine, valine, alanine, leucine or isoleucine. In anotherembodiment, C consists of a sequence of one to three amino acids,selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys,Cit, Ser, and Glu. More preferably, it is a two to three amino acidpeptide from the foregoing group.

The preparation and design of cleavable groups C consisting of a singleamino acid is disclosed in Chen et al., U.S. Pat. No. 8,664,407 B2(2014), the disclosure of which is incorporated herein by reference.

Group C can be bonded directly to Z or D; i.e. spacers X^(Z) or X^(D),as the case may be, can be absent.

When present, spacer X^(Z) provides spatial separation between C and Z,lest the former sterically interfere with antigen binding by latter orthe latter sterically interfere with cleavage of the former. Further,spacer X^(Z) can be used to confer increased solubility or decreasedaggregation properties to conjugates. A spacer X^(Z) can comprise one ormore modular segments, which can be assembled in any number ofcombinations. Examples of suitable segments for a spacer X^(Z) are:

and combinations thereof,where the subscript g is 0 or 1 and the subscript h is 1 to 24,preferably 2 to 4. These segments can be combined, such as illustratedbelow:

Spacer X^(D), if present, provides spatial separation between C and D,lest the latter interfere sterically or electronically with cleavage ofthe former. Spacer X^(D) also can serve to introduce additionalmolecular mass and chemical functionality into a conjugate. Generally,the additional mass and functionality will affect the serum half-lifeand other properties of the conjugate. Thus, through judicious selectionof spacer groups, the serum half-live of a conjugate can be modulated.Spacer X^(D) also can be assembled from modular segments, analogously tothe description above for spacer X^(Z).

Spacers X^(Z) and/or X^(D), where present, preferably provide a linearseparation of from 4 to 25 atoms, more preferably from 4 to 20 atoms,between Z and C or D and C, respectively.

The linker can perform other functions in addition to covalently linkingthe antibody and the drug. For instance, the linker can contain apoly(ethylene glycol) (“PEG”) group. Since the conjugation steptypically involves coupling a drug-linker to an antibody in an aqueousmedium, a PEG group many enhance the aqueous solubility of thedrug-linker. Also, a PEG group may enhance the solubility or reduceaggregation in the resulting ADC. Where a PEG group is present, it maybe incorporated into either spacer X^(Z) of X^(D), or both. The numberof repeat units in a PEG group can be from 2 to 20, preferably between 4and 10.

Either spacer X^(Z) or X^(D), or both, can comprise a self-immolatingmoiety. A self-immolating moiety is a moiety that (1) is bonded to C andeither Z or D and (2) has a structure such that cleavage from group Cinitiates a reaction sequence resulting in the self-immolating moietydisbonding itself from Z or D, as the case may be. In other words,reaction at a site distal from Z or D (cleavage from group C) causes theX^(Z)—Z or the X^(D)—D bond to rupture as well. The presence of aself-immolating moiety is desirable in the case of spacer X^(D) because,if, after cleavage of the conjugate, spacer X^(D) or a portion thereofwere to remain attached to D, the biological activity of D may beimpaired. The use of a self-immolating moiety is especially desirablewhere cleavable group C is a polypeptide, in which instance theself-immolating moiety typically is located adjacent thereto, in orderto prevent D from sterically or electronically interfering with peptidecleavage.

Exemplary self-immolating moieties (i)-(v) bonded to a hydroxyl or aminogroup of D are shown below:

The self-immolating moiety is the structure between dotted lines a and b(or dotted lines b and c), with adjacent structural features shown toprovide context. Self-immolating moieties (i) and (v) are bonded to aD-NH₂ (i.e., conjugation is via an amino group), while self-immolatingmoieties (ii), (iii), and (iv) are bonded to a D-OH (i.e., conjugationis via a hydroxyl or carboxyl group). Cleavage of the bond at dottedline b by an enzyme—a peptidase in the instance of structures (i)-(v)and a 0-glucuronidase in the instance of structure (vi)—initiates aself-immolating reaction sequence that results in the cleavage of thebond at dotted line a and the consequent release of D-OH or D-NH₂, asthe case may be. By way of illustration, self-immolating mechanisms forstructures (i) and (iv) are shown below:

In other words, cleavage of a first chemical bond at one part of aself-immolating group initiates a sequence of steps that results in thecleavage of a second chemical bond—the one connecting theself-immolating group to the drug—at a different part of theself-immolating group, thereby releasing the drug.

In some instances, self-immolating groups can be used in tandem, asshown by structure (vii). In such case, cleavage at dotted line ctriggers self-immolation of the moiety between dotted lines b and c by a1,6-elimination reaction, followed by self-immolation of the moietybetween dotted lines a and b by a cyclization-elimination reaction. Foradditional disclosures regarding self-immolating moieties, see Carl etal., J. Med. Chem. 1981, 24, 479; Carl et al., WO 81/01145 (1981);Dubowchik et al., Pharmacology & Therapeutics 1999, 83, 67; Firestone etal., U.S. Pat. No. 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 2002,67, 1866; Doronina et al., Nature Biotechnology 2003, 21, 778 (erratum,p. 941); Boyd et al., U.S. Pat. No. 7,691,962 B2; Boyd et al., US2008/0279868 A1; Sufi et al., WO 2008/083312 A2; Feng, U.S. Pat. No.7,375,078 B2; Jeffrey et al., U.S. Pat. No. 8,039,273; and Senter etal., US 2003/0096743 A1; the disclosures of which are incorporated byreference.

In another embodiment, Z and D are linked by a non-cleavable linker,i.e., C is absent. Metabolism of D eventually reduces the linker to asmall appended moiety that does not interfere with the biologicalactivity of D.

Conjugation Techniques

Conjugates of TLR7 agonists disclosed herein preferably are made byfirst preparing a compound comprising D and linker(X^(D))_(a)(C)_(c)(X^(Z))_(b) (where X^(D), C, X^(Z), a, b, and c are asdefined for formula (II)) to form drug-linker compound represented byformula (V):

D-(X^(D))_(a)(C)_(c)(X^(Z))_(b)—R³¹  (V)

where R³¹ is a functional group suitable for reacting with acomplementary functional group on Z to form the conjugate. Examples ofsuitable groups R³¹ include amino, azide, thiol, cyclooctyne,

where R³² is Cl, Br, F, mesylate, or tosylate and R³³ is Cl, Br, I, F,OH, —O—N-succinimidyl, —O-(4-nitrophenyl), —O-pentafluorophenyl, or—O-tetrafluorophenyl. Chemistry generally usable for the preparation ofsuitable moieties D-(X^(D))_(a)C(X^(Z))_(b)—R³¹ is disclosed in Ng etal., U.S. Pat. No. 7,087,600 B2 (2006); Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., U.S. Pat. No. 7,129,261 B2 (2006); Ng etal., WO 02/096910 A1; Boyd et al., U.S. Pat. No. 7,691,962 B2; Chen etal., U.S. Pat. No. 7,517,903 B2 (2009); Gangwar et al., U.S. Pat. No.7,714,016 B2 (2010); Boyd et al., US 2008/0279868 A1; Gangwar et al.,U.S. Pat. No. 7,847,105 B2 (2010); Gangwar et al., U.S. Pat. No.7,968,586 B2 (2011); Sufi et al., U.S. Pat. No. 8,461,117 B2 (2013); andChen et al., U.S. Pat. No. 8,664,407 B2 (2014); the disclosures of whichare incorporated herein by reference.

Preferably reactive functional group —R³¹ is —NH₂, —OH, —CO₂H, —SH,maleimido, cyclooctyne, azido (—N₃), hydroxylamino (—ONH₂) orN-hydroxysuccinimido. Especially preferred functional groups —R³¹ are:

An —OH group can be esterified with a carboxy group on the antibody, forexample, on an aspartic or glutamic acid side chain.

A —CO₂H group can be esterified with a —OH group or amidated with anamino group (for example on a lysine side chain) on the antibody.

An N-hydroxysuccinimide group is functionally an activated carboxylgroup and can conveniently be amidated by reaction with an amino group(e.g., from lysine).

A maleimide group can be conjugated with an —SH group on the antibody(e.g., from cysteine or from the chemical modification of the antibodyto introduce a sulfhydryl functionality), in a Michael additionreaction.

Where an antibody does not have a cysteine —SH available forconjugation, an ε-amino group in the side chain of a lysine residue canbe reacted with 2-iminothiolane orN-succinimidyl-3-(2-pyridyldithio)propionate (“SPDP”) to introduce afree thiol (—SH) group—creating a cysteine surrogate, as it were. Thethiol group can react with a maleimide or other nucleophile acceptorgroup to effect conjugation. The mechanism if illustrated below with2-iminothiolane.

Typically, a thiolation level of two to three thiols per antibody isachieved. For a representative procedure, see Cong et al., U.S. Pat. No.8,980,824 B2 (2015), the disclosure of which is incorporated herein byreference.

In a reversed arrangement, an antibody Z can be modified withN-succinimidyl 4-(maleimidomethyl)-cyclohexanecarboxylate (“SMCC”) orits sulfonated variant sulfo-SMCC, both of which are available fromSigma-Aldrich, to introduce a maleimide group thereto. Then, conjugationcan be effected with a drug-linker compound having an —SH group on thelinker.

An alternative conjugation method employs copper-free “click chemistry,”in which an azide group adds across a strained cyclooctyne to form an1,2,3-triazole ring. See, e.g., Agard et al., J Amer. Chem. Soc. 2004,126, 15046; Best, Biochemistry 2009, 48, 6571, the disclosures of whichare incorporated herein by reference. The azide can be located on theantibody and the cyclooctyne on the drug-linker moiety, or vice-versa. Apreferred cyclooctyne group is dibenzocyclooctyne (DIBO). Variousreagents having a DIBO group are available from Invitrogen/MolecularProbes, Eugene, Oreg. The reaction below illustrates click chemistryconjugation for the instance in which the DIBO group is attached to theantibody (Ab):

Yet another conjugation technique involves introducing a non-naturalamino acid into an antibody, with the non-natural amino acid providing afunctionality for conjugation with a reactive functional group in thedrug moiety. For instance, the non-natural amino acidp-acetylphenylalanine can be incorporated into an antibody or otherpolypeptide, as taught in Tian et al., WO 2008/030612 A2 (2008). Theketone group in p-acetylphenyalanine can be a conjugation site via theformation of an oxime with a hydroxylamino group on the linker-drugmoiety. Alternatively, the non-natural amino acid p-azidophenylalaninecan be incorporated into an antibody to provide an azide functionalgroup for conjugation via click chemistry, as discussed above.Non-natural amino acids can also be incorporated into an antibody orother polypeptide using cell-free methods, as taught in Goerke et al.,US 2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009,102 (2), 400-416. The foregoing disclosures are incorporated herein byreference. Thus, in one embodiment, an antibody that is used for makinga conjugate has one or more amino acids replaced by a non-natural aminoacid, which preferably is p-acetylphenylalanine or p-azidophenylalanine,more preferably p-acetylphenylalanine.

Still another conjugation technique uses the enzyme transglutaminase(preferably bacterial transglutaminase from Streptomyces mobaraensis orBTG), per Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995. BTG formsan amide bond between the side chain carboxamide of a glutamine (theamine acceptor) and an alkyleneamino group (the amine donor), which canbe, for example, the ε-amino group of a lysine or a 5-amino-n-pentylgroup. In a typical conjugation reaction, the glutamine residue islocated on the antibody, while the alkyleneamino group is located on thelinker-drug moiety, as shown below:

The positioning of a glutamine residue on a polypeptide chain has alarge effect on its susceptibility to BTG mediated transamidation. Noneof the glutamine residues on an antibody are normally BTG substrates.However, if the antibody is deglycosylated—the glycosylation site beingasparagine 297 (N297; numbering per EU index as set forth in Kabat etal., “Sequences of proteins of immunological interest,” 5th ed., Pub.No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md.,1991; hereinafter “Kabat”) of the heavy chain—nearby glutamine 295(Q295) is rendered BTG susceptible. An antibody can be deglycosylatedenzymatically by treatment with PNGase F (Peptide-N-Glycosidase F).Alternatively, an antibody can be synthesized glycoside free byintroducing an N297A mutation in the constant region, to eliminate theN297 glycosylation site. Further, it has been shown that an N297Qsubstitution not only eliminates glycosylation, but also introduces asecond glutamine residue (at position 297) that too is an amineacceptor. Thus, in one embodiment, the antibody is deglycosylated. Inanother embodiment, the antibody has an N297Q substitution. Thoseskilled in the art will appreciate that deglycosylation bypost-synthesis modification or by introducing an N297A mutationgenerates two BTG-reactive glutamine residues per antibody (one perheavy chain, at position 295), while an antibody with an N297Qsubstitution will have four BTG-reactive glutamine residues (two perheavy chain, at positions 295 and 297).

An antibody can also be rendered susceptible to BTG-mediated conjugationby introducing into it a glutamine containing peptide, or “tag,” astaught, for example, in Pons et al., US 2013/0230543 A1 (2013) andRao-Naik et al., WO 2016/144608 A1.

In a complementary approach, the substrate specificity of BTG can bealtered by varying its amino acid sequence, such that it becomes capableof reacting with glutamine 295 in an umodified antibody, as taught inRao-Naik et al., WO 2017/059158 A1 (2017).

While the most commonly available bacterial transglutaminase is thatfrom S. mobaraensis, transglutaminase from other bacteria, havingsomewhat different substrate specificities, can be considered, such astransglutaminase from Streptoverticillium ladakanum (Hu et al., US2009/0318349 A1 (2009), US 2010/0099610 A1 (2010), and US 2010/0087371A1 (2010)).

TLR7 agonists of this disclosure having a primary or secondary alkylamine are particularly suitable for use in conjugates, as the secondaryamine provides a functional group for attachment of the linker. Anexample of such a TLR7 agonist-linker compound is compound 41, whichcontains an enzymatically cleavable linker. FIG. 15 shows a schemeaccording to which compound 41 can be prepared.

An example of a TLR7 agonist-linker compound that contains anon-enzymatically cleavable linker is compound 43. FIG. 16 shows apathway for synthesizing compound 43.

Both compounds 41 and 43 contain a primary alkylamino group, renderingthem amenable to conjugation with transglutaminase. A suitableconjugation procedure is described in the Examples hereinbelow.

Conjugation can also be effected using the enzyme Sortase A, as taughtin Levary et al., PLoS One 2011, 6(4), e18342; Proft, Biotechnol. Lett.2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010); and Mao et al.,WO 2005/051976 A2 (2005). The Sortase A recognition motif (typicallyLPXTG, where X is any natural amino acid) may be located on the ligand Zand the nucleophilic acceptor motif (typically GGG) may be the group R³¹in formula (III), or vice-versa.

TLR7 Agonist Conjugates

Applying the fore-described techniques, TLR7 agonist conjugates such asthe ones shown below can be prepared:

where m is 1, 2, 3, or 4 and Ab is an antibody.

PEGylation

Attachment of a poly(ethylene glycol) (PEG) chain to a drug(“PEGylation”) can improve the latter's pharmacokinetic properties. Thecirculation half-life of the drug is increased, sometimes by over anorder of magnitude, concomitantly reducing the dosage needed to achievea desired therapeutic effect. PEGylation can also decrease metabolicdegradation of a drug and reduce its immunogenicity. For a review, seeKolate et al., J. Controlled Release 2014, 192, 167.

Initially, PEGylation was applied to biologic drugs. As of 2016, overten PEGylated biologics had been approved. Turecek et al., J.Pharmaceutical Sci. 2016, 105, 460. More recently, stimulated by thesuccessful application of the concept to biologics, attention has turnedtowards its application to small molecule drugs. In addition to theaforementioned benefits, PEGylated small molecule drugs may haveincreased solubility and cause fewer toxic effects. Li et al. Prog.Polymer Sci. 2013, 38, 421.

The compounds disclosed herein can be PEGylated. Where a compound has analiphatic primary or secondary amine or an aliphatic hydroxyl, such asthe case of the compounds shown below (arrows), it can be PEGylated viaan ester, amide, carbonate, or carbamate group with a carboxy-containingPEG molecule utilizing conventional techniques such asdicyclohexylcarbodiimide, HATU, N-hydroxysuccinimide esters, and thelike. Various other methods for PEGylating pharmaceutical molecules aredisclosed in Alconcel et al., Polymer Chem. 2011, 2, 1442, thedisclosure of which is incorporated herein by reference.

If desired, a TLR7 agonist disclosed herein can be PEGylated via anenzymatically cleavable linker comprising a self-immolating moiety, toallow release of the un-PEGylated agonist in a designed manner. Further,PEGylation can be combined with conjugation to a protein such as anantibody, if the PEG-containing molecule has a suitable functional groupsuch as an amine for attachment to the protein. The protein can providean additional therapeutic function or, if an antibody, can provide atargeting function. These concepts are illustrated in the followingreaction sequence, where TLR7-NH-R generically represents a TLR7agonist:

In the above reaction sequence, the valine-citrulline (Val-Cit)dipeptide is cleavable by the enzyme cathepsin B, with a p-aminobenzyloxycarbonyl (PABC) group serving as a self-immolating spacer. Thefunctional group for conjugation is an amine group, which is temporarilyprotected by an Fmoc group. Conjugation is effected by the enzymetransglutaminase, with a glutamine (Gln) side chain acting as the acylacceptor. The subscript x, denoting the number of PEG repeat units, canvary widely, depending on the purpose of the PEGylation, as discussedbelow. For some purposes, x can be relatively small, such as 2, 4, 8,12, or 24. For other purposes, x is large, for example between about 45and about 910.

Those skilled in the art will understand that the sequence isillustrative and that other elements—peptide, self-immolating group,conjugation method, PEG length, etc.—may be employed, as is well knownin the art. They will also understand that, while the above sequencecombines PEGylation and conjugation, PEGylation does not requireconjugation, and vice-versa.

Where the compound lacks aliphatic hydroxyl or aliphatic primary orsecondary amine, it still can be PEGylated at the aromatic amine on thepyrimidine ring. A method for PEGylating at this position is disclosedby Zarraga, US 2017/0166384 A1 (2007), the disclosure of which isincorporated by reference.

In some embodiments, it may be desirable to have multiple PEGylatedagonists linked in a single molecule. For instance, four PEGylated armscan be constructed on pentaerythritol (C(CH₂OH)₄) and a TLR7 agonist canbe attached to each PEGylated arm. See Gao et al., US 2013/0028857 A1(2013), the disclosure of which is incorporated by reference.

For modulating pharmacokinetics, it is generally preferred that the PEGmoiety have a formula weight of between about 2 kDa (corresponding toabout 45 —(CH₂CH₂O)— repeating units) and between about 40 kDa(corresponding to about 910 —(CH₂CH₂O)— repeating units), morepreferably between about 5 kDa and about 20 kDa. That is, the range ofthe subscript x in the above formulae is from about 45 to about 910. Itis to be understood that PEG compositions are not 100% homogeneous but,rather, exhibit a distribution of molecular weights. Thus, a referenceto, for example, “20 kDa PEG” means PEG having an average molecularweight of 20 kDa.

PEGylation can also be used for improving the solubility of an agonist.In such instances a shorter PEG chain can be used, for examplecomprising 2, 4, 8, 12, or 24 repeating units.

Pharmaceutical Compositions and Administration

In another aspect, there is provided a pharmaceutical compositioncomprising a compound of as disclosed herein, or of a conjugate thereof,formulated together with a pharmaceutically acceptable carrier orexcipient. It may optionally contain one or more additionalpharmaceutically active ingredients, such as a biologic or a smallmolecule drug. The pharmaceutical compositions can be administered in acombination therapy with another therapeutic agent, especially ananti-cancer agent.

The pharmaceutical composition may comprise one or more excipients.Excipients that may be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

Preferably, a pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound may be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrastemal injection and infusion. Alternatively, the pharmaceuticalcomposition can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to achievehigh drug concentration. The compositions can also be provided in theform of lyophilates, for reconstitution in water prior toadministration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 0.01 percent to about ninety-nine percent of activeingredient, preferably from about 0.1 percent to about 70 percent, mostpreferably from about 1 percent to about 30 percent of active ingredientin combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide a therapeutic response. Forexample, a single bolus may be administered, several divided doses maybe administered over time, or the dose may be proportionally reduced orincreased as indicated by the exigencies of the situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” refers to physically discrete units suited as unitary dosagesfor the subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic response, in association with the required pharmaceuticalcarrier.

The dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg, of the host body weight. For example dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-10 mg/kg,or alternatively 0.1 to 5 mg/kg. Exemplary treatment regimens areadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months, or onceevery three to 6 months. Preferred dosage regimens include 1 mg/kg bodyweight or 3 mg/kg body weight via intravenous administration, using oneof the following dosing schedules: (i) every four weeks for six dosages,then every three months; (ii) every three weeks; (iii) 3 mg/kg bodyweight once followed by 1 mg/kg body weight every three weeks. In somemethods, dosage is adjusted to achieve a plasma antibody concentrationof about 1-1000 μg/mL and in some methods about 25-300 μg/mL.

A “therapeutically effective amount” of a compound of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.For example, for the treatment of tumor-bearing subjects, a“therapeutically effective amount” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic compound can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human but can beanother mammal. Where two or more therapeutic agents are administered ina combination treatment, “therapeutically effective amount” refers tothe efficacy of the combination as a whole, and not each agentindividually.

The pharmaceutical composition can be a controlled or sustained releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,poly-glycolic acid, collagen, polyorthoesters, and polylactic acid. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices; (2) micro-infusion pumps;(3) transdermal devices; (4) infusion devices; and (5) osmotic devices.

In certain embodiments, the pharmaceutical composition can be formulatedto ensure proper distribution in vivo. For example, to ensure that thetherapeutic compounds of the invention cross the blood-brain barrier,they can be formulated in liposomes, which may additionally comprisetargeting moieties to enhance selective transport to specific cells ororgans.

INDUSTRIAL APPLICABILITY

TLR7 agonist compounds disclosed herein can be used for the treatment ofa disease or condition that can be ameliorated by activation of TLR7.

In one embodiment, the TLR7 agonist is used in combination with ananti-cancer immunotherapy agent—also known as an immuno-oncology agent.An anti-cancer immunotherapy agent works by stimulating a body's immunesystem to attack and destroy cancer cells, especially through theactivation of T cells. The immune system has numerous checkpoint(regulatory) molecules, to help maintain a balance between its attackinglegitimate target cells and preventing it from attacking healthy, normalcells. Some are stimulators (up-regulators), meaning that theirengagement promotes T cell activation and enhances the immune response.Others are inhibitors (down-regulators or brakes), meaning that theirengagement inhibits T cell activation and abates the immune response.Binding of an agonistic immunotherapy agent to a stimulatory checkpointmolecule can lead to the latter's activation and an enhanced immuneresponse against cancer cells. Reciprocally, binding of an antagonisticimmunotherapy agent to an inhibitory checkpoint molecule can preventdown-regulation of the immune system by the latter and help maintain avigorous response against cancer cells. Examples of stimulatorycheckpoint molecules are B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS,CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.Examples of inhibitory checkpoint molecules are CTLA-4, PD-1, PD-L1,PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1,CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, CD96 andTIM-4.

Whichever the mode of action of an anti-cancer immunotherapy agent, itseffectiveness can be increased by a general up-regulation of the immunesystem, such as by the activation of TLR7. Thus, in one embodiment, thisspecification provides a method of treating a cancer, comprisingadministering to a patient suffering from such cancer a therapeuticallyeffective combination of an anti-cancer immunotherapy agent and a TLR7agonist as disclosed herein. The timing of administration can besimultaneous, sequential, or alternating. The mode of administration cansystemic or local. The TLR7 agonist can be delivered in a targetedmanner, via a conjugate.

Cancers that could be treated by a combination treatment as describedabove include acute myeloid leukemia, adrenocortical carcinoma, Kaposisarcoma, lymphoma, anal cancer, appendix cancer, teratoid/rhabdoidtumor, basal cell carcinoma, bile duct cancer, bladder cancer, bonecancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor,cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia,chronic myeloproliferative neoplasm, colon cancer, colorectal cancer,craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma,esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, eye cancer,fallopian tube cancer, gallbladder cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumor, germ cell tumor, hairy cellleukemia, head and neck cancer, heart cancer, liver cancer,hypopharngeal cancer, pancreatic cancer, kidney cancer, laryngealcancer, chronic myelogenous leukemia, lip and oral cavity cancer, lungcancer, melanoma, Merkel cell carcinoma, mesothelioma, mouth cancer,oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngealcancer, prostate cancer, rectal cancer, salivary gland cancer, skincancer, small intestine cancer, soft tissue sarcoma, testicular cancer,throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginalcancer, and vulvar cancer.

Anti-cancer immunotherapy agents that can be used in combinationtherapies as disclosed herein include: AMG 557, AMP-224, atezolizumab,avelumab, BMS 936559, cemiplimab, CP-870893, dacetuzumab, durvalumab,enoblituzumab, galiximab, IMP321, ipilimumab, lucatumumab, MEDI-570,MEDI-6383, MEDI-6469, muromonab-CD3, nivolumab, pembrolizumab,pidilizumab, spartalizumab, tremelimumab, urelumab, utomilumab,varlilumab, vonlerolizumab. Table A below lists their alternativename(s) (brand name, former name, research code, or synonym) and therespective target checkpoint molecule.

TABLE A Immunotherapy Agent Alternative Name (s) Target AMG 557 B7RP-1(ICOSL) AMP-224 PD-1 Atezolizumab MPDL3280A, RO5541267, PD-L1TECENTRIQ ® Avelumab BAVENCIO ® PD-L1 BMS 936559 PD-L1 CemiplimabLIBTAYO ® PD-1 CP-870893 CD40 Dacetuzumab CD40 Durvalumab IMFINZI ®PD-L1 Enoblituzumab MGA271 B7-H3 Galiximab B7-1 (CD80) IMP321 LAG-3Ipilimumab YERVOY ® CTLA-4 Lucatumumab CD40 MEDI-570 ICOS (CD278)MEDI-6383 OX40 MEDI-6469 OX40 Muromonab-CD3 CD3 Nivolumab OPDIVO ® PD-1Pembrolizumab KEYTRUDA ® PD-1 Pidilizumab MDV9300 PD-1 SpartalizumabPDR001 PD-1 Tremelimumab Ticilimumab, CP-675, CTLA-4 CP-675,206 UrelumabBMS-663513 CD137 Utomilumab PF-05082566 CD137 Varlilumab CDX 1127 CD27Vonlerolizumab RG7888, MOXR0916, OX40 pogalizumab

In one embodiment of a combination treatment with a TLR7 agonist, theanti-cancer immunotherapy agent is an antagonistic anti-CTLA-4,anti-PD-1, or anti-PD-L1 antibody. The cancer can be lung cancer(including non-small cell lung cancer), pancreatic cancer, kidneycancer, head and neck cancer, lymphoma (including Hodgkin's lymphoma),skin cancer (including melanoma and Merkel skin cancer), urothelialcancer (including bladder cancer), gastric cancer, hepatocellularcancer, or colorectal cancer.

In another embodiment of a combination treatment with a TLR7 agonist,the anti-cancer immunotherapy agent is an antagonistic anti-CTLA-4antibody, preferably ipilimumab.

In another embodiment of a combination treatment with a TLR7 agonist,the anti-cancer immunotherapy agent is an antagonistic anti-PD-1antibody, preferably nivolumab or pembrolizumab.

The TLR7 agonists disclosed herein also are useful as vaccine adjuvants.

Biological Activity

The biological activity of compounds disclosed herein as TLR7 agonistscan be assayed by the procedures following.

Human TLR7 Agonist Activity Assay

This procedure describes a method for assaying human TLR7 (hTLR7)agonist activity of the compounds disclosed in this specification.

Engineered human embryonic kidney blue cells (HEK-Blue™ TLR cells;Invivogen) possessing a human TLR7-secreted embryonic alkalinephosphatase (SEAP) reporter transgene were suspended in a non-selective,culture medium (DMEM high-glucose (Invitrogen), supplemented with 10%fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to eachwell of a 384-well tissue-culture plate (15,000 cells per well) andincubated 16-18 h at 37° C., 5% CO₂. Compounds (100 nl) were dispensedinto wells containing the HEK-Blue™ TLR cells and the treated cells wereincubated at 37° C., 5% CO₂. After 18 h treatment ten microliters offreshly-prepared Quanti-Blue™ reagent (Invivogen) was added to eachwell, incubated for 30 min (37° C., 5% CO₂) and SEAP levels measuredusing an Envision plate reader (OD=620 nm). The half maximal effectiveconcentration values (EC₅₀; compound concentration which induced aresponse halfway between the assay baseline and maximum) werecalculated.

Induction of Type I Interferon Genes (MX-1) and CD69 in Human Blood

The induction of Type I interferon (IFN) MX-1 genes and the B-cellactivation marker CD69 are downstream events that occur upon activationof the TLR7 pathway. The following is a human whole blood assay thatmeasures their induction in response to a TLR7 agonist.

Heparinized human whole blood was harvested from human subjects andtreated with test TLR7 agonist compounds at 1 mM. The blood was dilutedwith RPMI 1640 media and Echo was used to predot 10 nL per well giving afinal concentration of 1 uM (10 nL in 10 uL of blood). After mixing on ashaker for 30 sec, the plates were covered and placed in a 37° C.chamber for o/n=17 hrs. Fixing/lysis buffer was prepared (5×->1× in H₂0,warm at 37° C.; Cat # BD 558049) and kept the perm buffer (on ice) forlater use.

For surface markers staining (CD69): prepared surface Abs: 0.045 ulhCD14-FITC (ThermoFisher Cat # MHCD1401)+0.6 ul hCD19-ef450(ThermoFisher Cat #48-0198-42)+1.5 ul hCD69-PE (cat # BD555531)+0.855 ulFACS buffer. Added 3 ul/well, spin 1000 rpm for 1 min and mixed onshaker for 30 sec, put on ice for 30 mins. Stop stimulation after 30minutes with 70 uL of prewarmed 1× fix/lysis buffer and use Feliex mateto resuspend (15 times, change tips for each plate) and incubate at 37Cfor 10 minutes.

Centrifuge at 2000 rpm for 5 minutes aspirate with HCS plate washer, mixon shaker for 30 sec and then wash with 70 uL in dPBS and pelleted 2×s(2000 rpm for 5 min) and 50 ul wash in FACS buffer pelleted 1×s (2000rpm for 5 min). Mix on shaker for 30 sec. For Intracellular markersstaining (MX-1): Add 50 ul of BD Perm buffer III and mix on shaker for30 sec. Incubate on ice for 30 minutes (in the dark). Wash with 50 uL ofFACS buffer 2× (spin @2300 rpm×5 min after perm) followed by mixing onshaker for 30 sec. Resuspended in 20 ul of FACS buffer containing MX1antibody ( )(4812)-Alexa 647: Novus Biologicals #NBP2-43704AF647) 20 ulFACS bf+0.8 ul hIgG+0.04 ul MX-1. Spin 1000 rpm for 1 min, mix on shakerfor 30 se and the samples were incubated at RT in the dark for 45minutes followed by washing 2×FACS buffer (spin @2300 rpm×5 min afterperm). Resuspend 20 ul (35 uL total per well) of FACS buffer and coverwith foil and place in 4° C. to read the following day. Plates were readon iQuePlus. The results were loaded into toolset and IC50 curves aregenerated in curve master. The y-axis 100% is set to 1 uM of resiquimod.

Induction of TNF-Alpha and Type I IFN Response Genes in Mouse Blood

The induction of TNF-alpha and Type I IFN response genes are downstreamevents that occur upon activation of the TLR7 pathway. The following isan assay that measures their induction in whole mouse blood in responseto a TLR7 agonist.

Heparinized mouse whole blood was diluted with RPMI 1640 media withPen-Strep in the ratio of 5:4 (50 uL whole blood and 40 uL of media). Avolume of 90 uL of the diluted blood was transferred to wells of Falconflat bottom 96-well tissue culture plates, and the plates were incubatedat 4° C. for 1 h. Test compounds in 100% DMSO stocks were diluted20-fold in the same media for concentration response assays, and then 10uL of the diluted test compounds were added to the wells, so that thefinal DMSO concentration was 0.5%. Control wells received 10 uL mediacontaining 5% DMSO. The plates were then incubated at 37° C. in a 5% CO₂incubator for 17 h. Following the incubation, 100 uL of the culturemedium as added to each well. The plates were centrifuged and 130 uL ofsupernatant was removed for use in assays of TNFa production by ELISA(Invitrogen, Catalog Number 88-7324 by Thermo-Fisher Scientific). A 70uL volume of mRNA catcher lysis buffer (1×) with DTT from the InvitrogenmRNA Catcher Plus kit (Cat # K1570-02) was added to the remaining 70 uLsample in the well, and was mixed by pipetting up and down 5 times. Theplate was then shaken at room temperature for 5-10 min, followed byaddition of 2 uL of proteinase K (20 mg/mL) to each well. Plates werethen shaken for 15-20 min at RT. The plates were then stored at −80° C.until further processing.

The frozen samples were thawed and mRNA was extracted using theInvitrogen mRNA Catcher Plus kit (Cat # K1570-02) according to themanufacturer's instructions. Half yield of mRNA from RNA extraction wereused to synthesize cDNA in 20 μL reverse transcriptase reactions usingInvitrogen SuperScript IV VILO Master Mix (Cat #11756500). TaqMan®real-time PCR was performed using QuantStudio Real-Time PCR system fromThermoFisher (Applied Biosystems). All real-time PCR reactions were runin duplicate using commercial predesigned TaqMan assays for mouse IFIT1,IFIT3, MX1 and PPIA gene expression and TaqMan Master Mix. PPIA wasutilized as the housekeeping gene. The recommendations from themanufacturer were followed. All raw data (Ct) were normalized by averagehousekeeping gene (Ct) and then the comparative Ct (ΔΔCt) method wereutilized to quantify relative gene expression (RQ) for experimentalanalysis.

General Procedures

The following general procedures were used for liquid chromatography(preparative or analytical) and nuclear magnetic resonance.

Liquid Chromatography

Unless noted otherwise, the following general conditions were used forhigh pressure liquid chromatography (HPLC) purification or for liquidchromatography-mass spectrometry (LC-MS):

-   -   Preparative HPLC/MS conditions A-1: Column: XBridge C18, 200        mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:        water with 0.05% trifluoroacetic acid; Mobile Phase B: 95:5        acetonitrile: water with 0.05% trifluoroacetic acid; Gradient: a        0-minute hold at 0% B, 0-40% B over 20 minutes, then a 0-minute        hold at 100% B; Flow Rate: 20 mL/min; Column Temperature: 25° C.    -   Preparative HPLC/MS conditions A-2: Column: XBridge C18, 200        mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:        water with 10 mM ammonium acetate; Mobile Phase B: 95:5        acetonitrile: water with 10 mM ammonium acetate; Gradient: a        0-minute hold at 7% B, 7-47% B over 20 minutes, then a 0-minute        hold at 100% B; Flow Rate: 20 mL/min; Column Temperature: 25° C.    -   Preparative HPLC/MS conditions A-3: Column: XBridge Shield RP18        OBD Column, 19×150 mm, 5 μm; Mobile phase A: water with 0.05%        TFA; Mobile phase B acetonitrile with 0.05% TFA; Flow rate: 18.9        mL/min; Gradient: 0% B for 2 min, 0-35% B for 20 min, 100% B for        3 min; Detector, UV 254/210 nm; Column Temperature: 25° C.    -   LC/MS conditions B: Column: Aquity UPLC BEH C18, 2.1 mm×50 mm,        1.7 μm particles; Mobile Phase A: 100% water with 0.05% TFA;        Mobile Phase B: 100% acetonitrile with 0.05% TFA; Gradient: 2% B        to 98% B over 1 min, then a 0.50 min hold at 98% B; Flow: 0.8        mL/min.    -   LC/MS conditions C: Column: Waters XBridge C18, 2.1 mm×50 mm,        1.7 μm particles; Mobile Phase A: 5:95 acetonitrile:water with        0.1% trifluoroacetic acid; Mobile Phase B: 95:5        acetonitrile:water with 0.1% trifluoroacetic acid; Temperature:        50° C.; Gradient: 0% B to 100% B over 3 min, then a 0.50 min        hold at 100% B; Flow: 1 mL/min; Detection: MS and UV (220 nm).    -   LC/MS conditions D: LC/MS conditions: Column: Aquity UPLC BEH        C18, 2.1 mm×50 mm, 1.7 μm particles; Mobile Phase A: 95:5        water:acetonitrile with 10 mM NH₄OH; Mobile Phase B: 95:5        acetonitrile:water with 10 mM NH₄OH; Gradient: 5% B to 95% B        over 1 min, then a 0.50 min hold at 95% B; Flow: 0.8 mL/min to        95% B over 1 min, then a 0.50 min hold at 95% B; Flow: 0.8        mL/min.    -   LC/MS conditions E: LC/MS conditions: Column: Waters XBridge        C18, 2.1 mm×50 mm, 1.7 μm particles; Mobile Phase A: water with        0.1% TFA; Mobile Phase B: acetonitrile with 0.1% TFA;        Temperature: 40° C.; Gradient: 5% B to 95% B over 2.6 min, then        a 0.20 min hold at 95% B; Flow rate: 0.6 mL/min;

NMR

The following conditions were used for obtaining proton nuclear magneticresonance (NMR) spectra: NMR spectra were taken in either 400 Mz or 500Mhz Bruker instrument using either DMSO-d6 or CDCl₃ as solvent andinternal standard. The crude NMR data was analyzed by using either ACDSpectrus version 2015-01 by ADC Labs or MestReNova software.

Chemical shifts are reported in parts per million (ppm) downfield frominternal tetramethylsilane (TMS) or from the position of TMS inferred bythe deuterated NMR solvent. Apparent multiplicities are reported as:singlet-s, doublet-d, triplet-t, quartet-q, or multiplet-m. Peaks thatexhibit broadening are further denoted as br. Integrations areapproximate. It should be noted that integration intensities, peakshapes, chemical shifts and coupling constants can be dependent onsolvent, concentration, temperature, pH, and other factors. Further,peaks that overlap with or exchange with water or solvent peaks in theNMR spectrum may not provide reliable integration intensities. In somecases, NMR spectra may be obtained using water peak suppression, whichmay result in overlapping peaks not being visible or having alteredshape and/or integration.

Synthesis

The practice of this invention can be further understood by reference tothe following examples, which are provided by way of illustration andnot of limitation.

Generally, the procedures disclosed herein produce a mixture ofregioisomers, alkylated at the 1H or 2H position of thepyrazolopyrimidine ring system (which are also referred to as N1 and N2regioisomers, respectively, alluding to the nitrogen that is alkylated).In the figures, sometimes the N2 regioisomers are not shown forconvenience, but it is to be understood that they are present in theinitial product mixture and separated at a later time, for example bypreparative HPLC.

The mixture of regioisomers can be separated at an early stage of thesynthesis and the remaining synthetic steps carried out with the 1Hregioisomer or, alternatively, the synthesis can be progressed carryingthe mixture of regioisomers and separation effected at a later stage, asdesired.

Procedure 1—Compounds Per FIG. 1

This procedure and companion FIG. 1 illustrate a method for makingcompounds disclosed herein, using compound 800 as an exemplar.

Compound 3. Methyl 4-amino-1H-pyrazole-5-carboxylate 2 (4 g, 28.3 mmol)and methyl(Z)-4-(2,3-bis(methoxycarbonyl)guanidino)-1H-pyrazole-5-carboxylate 1 inmethanol (50 mL) was treated with acetic acid (8.11 mL, 142 mmol) atwhich time a precipitate formed. The reaction mixture was stirredovernight. Sodium methoxide (64.8 mL, 283 mmol) was added and stirringwas continued overnight. LCMS showed completion of the reaction. The pHwas adjusted to 5 by the slow addition of acetic acid, whereby aprecipitate formed that was washed with water and then acetonitrile anddried to provide 5.2 g of compound 3 as an off white solid. LCMS ESI:calculated for C₇H₇N₅O₃=210.16 (M+H⁺), found 210.0 (M+H⁺).

Compound 4. Compound 3 (2 g, 9.56 mmol), butan-1-amine (1.8 mL, 9 mmol),and DBU (1.6 mL, 10 mmol) in DMSO (10 mL) was slowly treated with BOP (5g, 11 mmol). The reaction mixture was heated at 60° C. for 2 h at whichtime LCMS showed completion of the reaction. The reaction was directlypurified on reverse phase COMBIFLASH™ apparatus using 80 g C-18 columneluting with 0-100% acetonitrile/water (0.1% formic acid) to yieldcompound 4 as a white solid. LCMS ESI: calculated for C₁₁H₁₆N₆O₂=265.28(M+H⁺), found 265.2 (M+H⁺). ¹H NMR (400 MHz, dmso-d6) δ 8.02 (s, 1H),3.97 (s, 3H), 1.74-1.66 (m, 2H), 1.49-1.38 (m, 2H), 1.25 (s, 1H), 0.95(t, J=7.4 Hz, 3H).

Compound 6. A mixture of methyl 4-(bromomethyl)-2-methoxybenzoate 5 (1g, 3.86 mmol) and cyclobutanamine 5a (0.659 mL, 7.72 mmol) in DMF (2 mL)was heated at 70° C. over 30 min at which point LCMS showed theformation of an amine product. The excess base was evaporated andHunig's Base (1.348 mL, 7.72 mmol) was added, followed by addition ofBoc-anhydride (0.896 mL, 3.86 mmol). LCMS showed the completion ofreaction. The solvent was evaporated and the crude product was purifiedby COMBIFLASH™ apparatus using EtOAc/hexanes to provide 0.82 g desiredproduct 6 as a colorless oil. LCMS ESI: calculated for C₁₉H₂₇NO₅=350.42(M+H⁺), found 350.1 (M+H⁺).

Compound 7. A solution of compound 6 (0.82 g, 2.347 mmol) in THF (5 mL)at 0° C. was treated slowly with LiAlH₄ (2 M in THF, 1.173 mL, 2.347mmol) and stirred for 30 min, at which point LCMS showed completion ofthe reaction. The reaction was quenched by the slow addition of methanoland stirred with Rochelle salt solution for 2 h. The organic layers wereseparated and the crude product 7 was purified on a COMBIFLASH™apparatus using EtOAc/hexanes, silica gel column. LCMS ESI: calculatedfor C₁₈H₂₇NO₄=322.41 (M+H⁺), found 322.1 (M+H⁺).

Compounds 8 and 9. A mixture of compound 4 (100 mg, 0.378 mmol),compound 7 (182 mg, 0.568 mmol) and triphenylphosphine (248 mg, 0.946mmol) in THF (3 mL) was slowly treated with DIAD (0.110 mL, 0.568 mmol)over 5 min and stirred at RT for 30 min under N₂ at which point LCMSshowed the completion of the reaction. The solvent was evaporated andthe crude product was purified on reverse phase COMBIFLASH™ apparatususing 80 g C-18 column eluting with 0-100% acetonitrile/water (1 mMTEAA) to provide a mixture of compounds 8 and 9 as a white solid. LCMSESI: calculated for C₂₉H₄₁N₇O₅=566.69 (M−H⁺), found 566.3 (M−H⁺).

The isomers were separated by chiral supercritical fluid chromatographyusing Column: Kromasil 5-CelluCoat, 21×250 mm, 5 micron, Mobile Phase:15% MeOH-DEA/85% CO₂, Flow Conditions: 45 mL/min, 150 Bar, 40° C.,Detector Wavelength: 230 nm, Injection Details: 0.5 mL of −25 mg/mL inMeOH to provide 17 mg of compound 8 and 25 mg of compound 9.

-   -   Analytical data for compound 8: ¹H NMR (400 MHz, DMSO-d6) δ 9.61        (s, 2H), 7.86 (s, 2H), 6.94 (s, 2H), 6.83 (s, 2H), 6.63 (d,        J=7.9 Hz, 2H), 6.51 (d, J=7.5 Hz, 2H), 5.69 (s, 4H), 4.39 (s,        4H), 3.79 (s, 6H), 3.63 (s, 6H), 3.48 (d, J=6.2 Hz, 4H), 3.33        (s, 20H), 3.18 (d, J=5.3 Hz, 1H), 2.05-1.95 (m, 8H), 1.53 (t,        J=7.5 Hz, 7H), 1.35 (s, 11H), 1.23 (q, J=7.2 Hz, 6H), 0.86 (t,        J=7.4 Hz, 6H).    -   Analytical data for compound 9: ¹H NMR (400 MHz, DMSO-d6) δ 9.37        (s, 1H), 8.08 (s, 1H), 6.90 (s, 1H), 6.84 (s, 1H), 6.71 (d,        J=7.7 Hz, 1H), 5.48 (s, 2H), 4.42 (s, 3H), 3.78 (d, J=18.0 Hz,        4H), 3.69 (s, 1H), 3.61 (s, 3H), 3.51-3.42 (m, 3H), 2.06-1.97        (m, 6H), 1.61-1.47 (m, 6H), 1.36 (s, 8H), 1.34-1.26 (m, 6H),        0.99 (t, J=7.1 Hz, 3H), 0.94-0.85 (m, 4H).

Compound 800. A solution of compound 8 (13 mg, 0.023 mmol) was dissolvedin THF (0.5 mL) and was treated with TFA (0.018 mL, 0.229 mmol). LCMS in30 min showed Boc deprotection. The TFA was evaporated and this mixturewas treated with sodium hydroxide (9.16 mg, 0.229 mmol) and heated at60° C. for 2 h, at which point LCMS showed completion of the reaction.The base was neutralized by the slow addition of 6M HCl and worked upwith EtOAc/water. The crude material was purified via preparative HPLCunder HPLC/MS conditions A-2.

Other compounds of this disclosure can be analogously made, mutatismutandis, by using another amine instead of cyclobutanamine 5a.

Procedure 2 Compounds Per FIGS. 2A-2B

This procedure and companion FIGS. 2A-2B illustrate another method formaking compounds disclosed herein, using compounds 801 and 818 asexemplars.

Compounds 12 and 13. A solution of methyl4-nitro-1H-pyrazole-5-carboxylate 10 (3.27 g, 19.11 mmol) in DMF (20 mL)was treated with K₂CO₃ (2.90 g, 21.02 mmol) and methyl4-(bromomethyl)-3-methoxybenzoate 11 (5 g, 19.30 mmol). The reaction wasstarted at 0° C. and allowed to proceed for 1 h, at which point LCMSshowed completion of the reaction with −1:5 mixture of products. Thebase was filtered and the reaction was diluted with EtOAc and washedwith water 2 times. The solvent was evaporated and the crude product wastaken to next step as-is. LCMS ESI: calculated for C₁₅H₁₅N₃O₇=350.2(M−H⁺), found 350.0 (M−H⁺).

For characterization purpose, a small amount of the mixture of productswas separated using silica gel column chromatography using 0-50%EtOAc/hexanes.

-   -   Analytical data for compound 12: ¹H NMR (400 MHz, DMSO-d6) δ        8.40 (s, 1H), 7.57 (dd, J=7.8, 1.5 Hz, 1H), 7.50 (d, J=1.6 Hz,        1H), 7.27 (d, J=7.9 Hz, 1H), 5.53 (s, 2H), 3.96 (s, 3H), 3.84        (d, J=16.2 Hz, 6H).    -   Analytical data for compound 13: ¹H NMR (400 MHz, DMSO-d6) δ        9.05 (s, 1H), 7.62-7.51 (m, 2H), 7.28 (d, J=7.9 Hz, 1H), 5.47        (s, 2H), 3.87 (s, 8H), 3.31 (s, 1H).

Compounds 14 and 15. A solution of compounds 12 and 13 (2 g, 5.73 mmol),zinc and ammonium formate was stirred at RT for 2 h, after which LCMSshowed completion of the reaction. Filtration and concentration yieldeda crude mixture of compounds 14 and 15. LCMS ESI: calculated forC₁₅H₁₇N₃O₅=320.3 (M+H⁺), found 320.2 (M+H⁺).

Compounds 16 and 17. A mixture of compounds 14 and 15 (1.830 g, 5.73mmol) and compound 1 in MeOH (20 mL) was treated with acetic acid (1.640mL, 28.7 mmol) and stirred overnight. The solution was treated withsodium methoxide (13.11 mL, 57.3 mmol) and stirred overnight. LCMSshowed conversion to the product. The pH was adjusted to 5 and theresulting precipitate was washed with water. The residue was dried toafford a mixture of compounds 16 and 17. LCMS ESI: calculated forC₁₇H₁₇N₅O₆=388.3 (M+H⁺), found 388.1 (M+H⁺).

Compounds 18 and 19. A mixture of compounds 16 and 17 (1 g, 2.58 mmol)in DMSO (10 mL) was treated with butan-1-amine (0.510 mL, 5.16 mmol),DBU (0.428 mL, 2.84 mmol) followed slowly by BOP (1.370 g, 3.10 mmol).The reaction was heated at 70° C. for 2 h, at which point LCMS showedcompletion of the reaction. The reaction was diluted with water andextracted with EtOAc. The combined organic phases were dried over Na₂SO₄and taken as-is to the next step. LCMS ESI: calculated forC₂₁H₂₆N₆O₅=443.4 (M+H⁺), found 443.2 (M+H⁺).

¹H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H),7.57-7.42 (m, 3H), 6.93 (d, J=8.1 Hz, 1H), 5.80 (s, 1H), 5.60 (s, 2H),4.04 (q, J=7.1 Hz, 1H), 3.95-3.82 (m, 10H), 3.62 (d, J=6.1 Hz, 4H), 3.45(q, J=7.0 Hz, 3H), 2.68 (d, J=9.9 Hz, 1H), 2.57-2.50 (m, 6H), 2.00 (s,1H), 1.59 (p, J=7.3 Hz, 3H), 1.54-1.48 (m, 1H), 1.39-1.26 (m, 3H), 1.18(t, J=7.1 Hz, 2H), 0.86 (dt, J=29.5, 7.3 Hz, 5H).

Compound 818. A solution of compounds 18 and 19 (1.142 g, 2.58 mmol) inTHF (2.58 mL, 5.16 mmol) at 0° C. was treated with LiAlH₄ (THF, 2.58 mL,5.16 mmol) and stirred for 1 h, after which LCMS showed completion ofthe reaction. The reaction was quenched with MeOH and stirred withRochelle salt solution overnight. The product was extracted with EtOAcand taken to next step as a mixture of crude compounds reducedintermediate. LCMS ESI: calculated for C₂₀H₂₆N₆O₄=415.4 (M+H⁺), found415.2 (M+H⁺).

A mixture of the reduced intermediates (1069 mg, 2.58 mmol) in1,4-dioxane (10 mL) was treated with aqueous sodium hydroxide (2.58 mL,25.8 mmol) and heated at 80° C. for 5 h, after which LCMS showedformation of product. The base was neutralized with 6M HCl and thesolvent was evaporated. The residue was taken up in 5 mL DMF and syringefiltered. The solvent was evaporated to afford a 3:1 mixture ofcompounds 818 and its regioisomer 19a.

Compound 801 and 19b. A solution of compounds 818 and 19a (420 mg, 1.178mmol) in THF (1 mL) was treated with thionyl chloride (0.172 mL, 2.357mmol) and stirred for 30 min, after which LCMS showed completion of thereaction. The solvent was evaporated and the crude benzyl chlorideintermediate was taken to next step as-is. LCMS ESI: calculated forC₁₈H₂₃ClN₆O=375.8 (M+H⁺), found 375.2 (M+H⁺).

A mixture of the preceding crude product mixture (20 mg, 0.053 mmol) andtetrahydro-2H-pyran-4-amine 22 (5.40 mg, 0.053 mmol) in DMF (1 mL) washeated at 70° C. for 1 h, after which LCMS showed completion of thereaction. The reaction was syringe filtered and the crude products werepurified and separated via preparative LC/MS with the followingconditions: Column: XBridge C18, 200 mm×19 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; MobilePhase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid;Gradient: a 2-minute hold at 6% B, 6-27% B over 25 minutes, then a2-minute hold at 100% B; Flow Rate: 20 mL/min; Column Temperature: 25 C.Fraction collection was triggered by MS signals. Fractions containingthe desired product were combined and dried via centrifugal evaporation.

If desired, the separation of 1H and 2H regioisomers can be effectedearlier in the synthetic process, for example at the mixture ofcompounds 12 and 13 and synthetic steps carried out only with the 1Hregioisomer.

Other compounds of this disclosure can be analogously prepared, mutatismutandis, for example by using another amine instead oftetrahydro-2H-pyran-4-amine 22.

Procedure 3—Compounds Per FIGS. 3A-3B

This procedure and companion FIGS. 3A-3B illustrate another method formaking compounds disclosed herein, using compounds 844, 817, and 861 asexemplars.

Compound 24. To a mixture of compound 16 (400 mg, 1.033 mmol) and(S)-3-aminohexan-1-ol 23 (242 mg, 2.065 mmol) in DMSO (5 mL) was addedDBU (0.463 mL, 3.10 mmol) followed by the slow addition of BOP (502 mg,1.136 mmol). The reaction mixture was heated at 70° C. for 2 h, at whichpoint LCMS showed completion of the reaction. The reaction mixture wasdiluted with EtOAc and washed with water. The organic layer wasconcentrated to leave a crude product, which was used in the next step.

The crude product was treated with tert-butylchlorodiphenylsilane (0.295mL, 1.136 mmol) and imidazole (141 mg, 2.065 mmol) in DMF (5 mL). Thereaction mixture was stirred overnight. The reaction mixture was dilutedwith water and worked up with EtOAc. Purification was performed with aCOMBIFLASH™ apparatus using EtOAc/hexane to afford compound 24. LCMSESI: calculated for C₃₈H₄₈N₆O₆Si=725.9 (M+H⁺), found 725.3 (M+H⁺). ¹HNMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.94 (s, 1H), 7.57-7.50 (m, 2H),7.50-7.30 (m, 7H), 7.25-7.14 (m, 3H), 6.31 (d, J=7.9 Hz, 1H), 6.24 (d,J=8.4 Hz, 1H), 5.78 (d, J=17.2 Hz, 2H), 4.56 (s, 1H), 3.92-3.82 (m, 4H),3.78 (s, 3H), 3.59 (s, 3H), 3.55-3.43 (m, 2H), 3.31 (s, 2H), 1.86-1.68(m, 2H), 1.47 (q, J=8.7, 7.9 Hz, 2H), 1.18 (t, J=7.1 Hz, 5H), 0.91 (s,9H).

Compound 25. A solution of compound 24 (408 mg, 0.563 mmol) in THF (5mL) was treated slowly with LiAlH₄ (0.563 mL, 1.126 mmol) and thereaction was stirred at 0° C. for 1 h, at which point LCMS showedcompletion of the reaction. The reaction was quenched with MeOH andstirred with Rochelle salt solution overnight. Extraction with EtOAc andpurification with a COMBIFLASH™ apparatus, 0-50% MeOH in DCM gradientafforded compound 25. LCMS ESI: calculated for C₃₈H₄₈N₆O₅Si: 697.9(M+H⁺), found 679.4 (M+H⁺).

Compound 26. A solution of compound 25 (150 mg, 0.215 mmol) in THF (0.5mL) was treated slowly with thionyl chloride (0.031 mL, 0.430 mmol) andstirred for 1 h. LCMS showed completion of the reaction. The solvent wasevaporated and the crude product 26 was taken to next step. LCMS ESI:calculated for C₃₈H₄₇ClN₆O₄Si=715.3 (M+H⁺), found 715.4 (M+H⁺).

Compound 844. Compound 26 (15 mg) was dissolved in DMF (0.5 mL) andtreated with 1,2,3,4-tetrahydro-2,6-naphthyridine 27 (5.78 mg, 0.043mmol) and heated at 70° C. for 2 h after which LCMS showed completion ofthe reaction. A solution of the intermediate product (0.018 g, 0.022mmol) was dissolved in dioxane (0.5 mL) and treated with triethylaminetrihydrofluoride (0.018 mL, 0.110 mmol). After stirring at RT overnight,LCMS showed removal of the TBDPS protecting group. Neutralization to pH7, evaporation of solvent and purification on a reverse phase ISCO usingTEAA as modifier afforded the deprotected intermediate product.

The deprotected intermediate product from above was dissolved in dioxane(0.5 mL), treated with sodium hydroxide (0.220 mL, 0.220 mmol) andheated at 80° C. for 2 h. LCMS showed completion of the reaction. Thereaction mixture was neutralized to pH 7 with aqueous HCl and thesolvent was evaporated. The residue was dissolved in DMF andsyringe-filtered and purified by preparative LC/MS using followingconditions: Column: XBridge C18, 200 mm×19 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; MobilePhase B: 95:5 acetonitrile: water with 10 mM ammonium acetate; Gradient:a 0-minute hold at 15% B, 15-55% B over 20 minutes, then a 4-minute holdat 100% B; Flow Rate: 20 mL/min; Column Temperature: 25° C. Fractioncollection was triggered by MS signals. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation to yieldcompound 844 (4.7 mg, 40% yield).

Compound 817. Compound 25 was treated with trimethylaminetrihydrofluoride and then sodium hydroxide, generally following theabove procedure, to afford compound 817.

FIG. 3B shows a variation of the scheme of FIG. 3A, in which the stepssubsequent to the preparation of compound 26 are varied. The scheme ofFIG. 3B is illustrated by particular reference to compound 861.

Compound 26b. Compound 26 (22 mg, 0.031 mmol) was dissolved in DMF (0.6mL) at RT and was treated with DIEA (27 μL, 0.15 mmol) and3-methoxyazetidine (26a) as its hydrochloride (11 mg, 0.093 mmol). Afterstirring for 2 h at 70° C., the reaction mixture was concentrated invacuo to give compound 26b, which was used without further purification.LC/MS conditions B: LC RT: 0.88 min. LCMS (M+H)=766.9.

Compound 861. A solution of compound 26a (24 mg, 0.031 mmol)) in dioxane(0.3 mL) at RT was treated with NaOH (10M aq. Solution, 0.1 mL, 1.0mmol) and the reaction was heated at 70° C. for 60 min, before theaddition of another portion of NaOH (10M aq. solution, 0.1 mL, 1.0mmol). After 2 h, the reaction mixture concentrated in vacuo. Theresidue was treated with MeOH (0.3 mL) and HCl (37% aq. solution, 0.3mL, 3.7 mmol), stirred at RT for 30 min and concentrated. The crudeproduct was dissolved in DMF, filtered through a PTFE frit, and purifiedvia preparative HPLC conditions A-1. Fraction collection was triggeredby MS signals. Fractions containing the desired product were combinedand dried via centrifugal evaporation to give compound 861 as its TFAsalt (8.4 mg, 46%). LC/MS conditions C: LC RT: 0.82 min. LCMS(M+H)=470.3.

Other compounds of this disclosure can be analogously prepared, mutatismutandis, for example by using amines other than those shown in FIGS.3A-3B.

Procedure 4 Compounds per FIGS. 4A-4B

This procedure and companion FIGS. 4A-4B illustrate another method ofmaking compounds disclosed herein.

Compound 30. A suspension of compound 4 (400 mg, 1.513 mmol) in dioxane(5 mL) was treated with sodium hydroxide (10 N in water, 1.513 mL, 15.13mmol) and stirred at 60° C. for 45 min. The reaction mixture wasconcentrated. The crude product was dissolved into water and purified byreverse phase chromatography on a COMBIFLASH™ unit using a 150 g C-18column eluting with 10 mM TEAA in acetonitrile: 10 mM in water, 0-70%gradient. The desired fractions were frozen and lyophilized to yieldcompound 30 (150 mg, 0.727 mmol, 48.1% yield). LCMS ESI: calculated forC₉H₁₅N₆=207.1 (M+H⁺), found 207.2 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ7.56 (br s, 1H), 5.53 (br s, 2H), 3.43 (br d, J=6.2 Hz, 2H), 1.57 (t,J=7.2 Hz, 2H), 1.44-1.29 (m, 2H), 0.95-0.76 (m, 3H).

Compound 33. A suspension of 6-fluoronicotinaldehyde 31 (1.809 g, 14.46mmol), methyl 4-hydroxybenzoate 32 (2 g, 13.15 mmol), and K₂CO₃ (1.998g, 14.46 mmol) in DMF (26.3 ml) was stirred at 110° C. for 4 h. LCMSindicated the reaction was complete. Upon cooling, the reaction wasquenched with water. The resulting solid was collected by filtration andrinsed with water and dried in vacuo to yield compound 33 (3.30 g, 12.84mmol, 95.1% yield). LCMS ESI: calculated for C₁₄H₁₁NO₄=258.1 (M+H⁺),found 258.0 (M+H⁺). ¹H NMR (400 MHz, CHLOROFORM-d) δ 10.01 (s, 1H), 8.63(d, J=2.4 Hz, 1H), 8.23 (dd, J=8.6, 2.4 Hz, 1H), 8.17-7.97 (m, 2H),7.27-7.22 (m, 2H), 7.10 (d, J=8.6 Hz, 1H), 3.93 (s, 3H).

Compound 34. A solution of compound 33 (3.76 g, 14.62 mmol) in MeOH (100ml) was treated with NaBH₄ (0.553 g, 14.62 mmol) portionwise at 0° C.and then stirred for 10 min with continued cooling. LCMS indicated thereaction was complete. Reaction was quenched by slowly adding halfsaturated NH₄Cl. Stirring was continued for 30 min at RT. The reactionmixture was extracted with ethyl acetate. The organic extracts weredried over Na₂SO₄, filtered, and concentrated. The crude solid wasslurried into water and collected by filtration and dried in vacuo toyield compound 34 (3.37 g, 13.00 mmol, 89% yield). LCMS ESI: calculatedfor C₁₄H₁₃NO₄=260.1 (M+H⁺), found 260.0 (M+H⁺). ¹H NMR (400 MHz,CHLOROFORM-d) δ 8.21 (d, J=2.2 Hz, 1H), 8.12-8.04 (m, 2H), 7.81 (dd,J=8.4, 2.4 Hz, 1H), 7.21-7.13 (m, 2H), 6.99 (d, J=8.4 Hz, 1H), 4.71 (s,2H), 3.91 (s, 3H).

Compound 35. Compound 34 (7.9 g, 30.5 mmol) in DCM (75 mL) was treatedwith MsCl (2.61 mL, 33.5 mmol) at 0° C. After stirring at RT for 16 h,the reaction was done. The reaction was quenched with water. Afterextraction with DCM (3×20 mL), the combined organic extracts were driedover Na₂SO₄, filtered and concentrated. The crude product was purifiedon an ISCO silica column (80 g), eluting with ethyl acetate:hexanes,0-70% gradient. The desired fractions were concentrated to yieldcompound 35 (7.47 g, 26.9 mmol, 88% yield). LCMS ESI: calculated forC₁₄H₁₃ClNO₃=278.1 (M+H⁺), found 278.0 (M+H⁺). 1H NMR (400 MHz,CHLOROFORM-d) δ 8.19 (d, J=2.2 Hz, 1H), 8.13-8.02 (m, 2H), 7.79 (dd,J=8.5, 2.5 Hz, 1H), 7.22-7.14 (m, 2H), 6.99 (d, J=8.6 Hz, 1H), 4.57 (s,2H), 3.92 (s, 3H).

Compounds 36 and 37. Compound 30 (70 mg, 0.339 mmol) in DMF (1 mL) wastreated with cesium carbonate (332 mg, 1.018 mmol), followed by compound35 (94 mg, 0.339 mmol). After stirring for 5 h at RT, the reaction wascomplete. After quenching with water and extraction with ethyl acetate(3×10 mL), the combined organic extracts were dried over Na₂SO₄,filtered and concentrated The crude product was purified on an ISCOsilica column (24 g), eluted with 20% MeOH in DCM:DCM, 0-60% gradient.The desired fractions were concentrated to yield a mixture of compounds36 and 37 (120 mg, 0.080 mmol, 79% yield), in a 1:4 ratio. LCMS ESI:calculated for C₂₃H₂₆N₇O₃=448.2 (M+H⁺), found 448.3 (M+H⁺).

Compounds 37a and 37b. A mixture of compounds 36 and 37 (60 mg, 0.114mmol) in THF (2 mL) was treated with LiAlH₄ (1.0 M in THF, 0.22 mL, 0.22mmol) slowly at 0° C. under N₂. The reaction mixture was stirred at 0°C. for 1 h, at which point LCMS showed completion of the reaction.Reaction was quenched by adding Na₂SO₄.10H₂0 slowly, followed by MeOHand stirring at RT for 3 h. The solid was filtered off. The filtrate wasconcentrated. The residue dissolved in DMF and the products werepurified via preparative LC/MS with the following conditions: Column:XBridge C18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: a 0-minutehold at 10% B, 10-45% B over 20 minutes, then a 4-minute hold at 100% B;Flow Rate: 20 mL/min. Fraction collection was triggered by MS and UVsignals. Fractions containing desired product were combined and driedvia centrifugal evaporation.

Compounds 38a and 38b. A mixture of compound 37a and compound 37b (40mg, 0.095 mmol) in THF (1 mL) was treated with thionyl chloride (0.14mL, 1.9 mmol). After stirring at RT for 3 h, LCMS showed completion ofthe reaction. The solvent was evaporated and the excess thionyl chloridewas azeotropically removed with DCM. The crude chloride material wasdirectly carried over to next step without further purification. LCMSESI: calculated for C₂₂H₂₅N₇O=438.2 (M+H⁺), found 438.1 (M+H⁺).

A mixture of the preceding chlorides (40 mg, 0.091 mmol) from precedingparagraph was dissolved in DMF (1.0 mL) and treated with2-aminoethan-1-ol (55.0 μl, 0.91 mmol), followed by stirring at RT for16 h. LCMS showed completion of the reaction. The mixture was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Gradient: a 0-minute hold at 8% B,8-48% B over 25 minutes, then a 4-min hold at 100% B; Flow Rate: 20mL/min; Column Temperature: 25° C. Fraction collection was triggered byMS signals. Fractions containing the desired product were combined anddried via centrifugal evaporation.

Replacing 6-fluoronicotinaldehyde 31 with 4-fluoro-2-methoxybenzaldehydein the scheme of FIGS. 4A-4B and generally following the proceduresabove, the other compounds of this disclosure can be prepared, forexample: 802, 803, 808, 809, and 810.

Procedure 5—Compounds Per FIG. 5

This procedure and companion FIG. 5 relate to the synthesis of(S)—N-(3-aminohexyl)acetamide 54, which can be used as an intermediatefor the synthesis of compounds disclosed herein.

Compound 51. A solution of (S)-3-aminohexan-1-ol 50 (1 g, 8.53 mmol) inDCM (10 mL) was treated with Hunig's base (4.47 mL, 25.6 mmol) andBoc-anhydride (2.377 mL, 10.24 mmol) and stirred for 3 h, after whichLCMS showed completion of the reaction. The solvent and base werestripped off in vacuo. The crude product was purified on an ISCO machinewith N₂ detector using 80 g silica gel gold column with using 0-100%EtOAc/hexanes to provide fractions with the desired product, which werecombined and concentrated to provide compound 51 (1.05 g, 56.6% yield).LCMS ESI: calculated for C₁₁H₂₃NO₃=218.3 (M+H⁺), found 218.2 ¹H NMR (400MHz, Chloroform-d) δ 3.76 (s, 1H), 3.63 (dd, J=7.6, 3.0 Hz, 2H),1.90-1.77 (m, 1H), 1.46-1.44 (m, 10H), 1.44-1.17 (m, 4H), 1.00-0.81 (m,5H).

Compound 52. To a solution of compound 51 (1050 mg, 4.83 mmol),triphenylphosphine (1648 mg, 6.28 mmol) and isoindoline-1,3-dione (924mg, 6.28 mmol) in THF (10 mL) was slowly added DIAD (1.221 mL, 6.28mmol). After stirring overnight at RT, LCMS showed completion of thereaction. Stripping off the solvent in a rotary evaporator andpurification on an ISCO machine using 80 g silical gold column elutingwith 0-50% EtOAc/hexanes provided fractions with the desired product,which were concentrated to provide compound 52 (1.6 g, 96% yield) as apale yellow solid. LCMS ESI: calculated for C₁₉H₂₆NO₄=347.4 (M+H⁺),found 247.2 (M-Boc+H⁺). δ 7.83 (dd, J=5.4, 3.1 Hz, 2H), 7.70 (dd, J=5.5,3.0 Hz, 2H), 4.40 (s, 1H), 3.75 (dd, J=8.4, 7.0 Hz, 2H), 3.64 (s, 1H),1.69 (dq, J=15.8, 7.9 Hz, 1H), 1.52-1.44 (m, 2H), 1.42 (s, 9H),1.39-1.28 (m, 3H), 0.91 (t, J=7.1 Hz, 3H).

Compound 53. Compound 52 (1.6 g, 4.62 mmol) was treated with methylaminein MeOH (15.59 g, 92 mmol). The reaction mixture was stirred for 1 h,after which LCMS showed phthalimide deprotection. The solvent and basewere evaporated and the residue was dissolved in THF (5 mL) and treatedwith Hunig's base (1.613 mL, 9.24 mmol), followed by acetyl chloride(0.394 mL, 5.54 mmol). The reaction mixture was stirred for 1 h at RTand the solvent was evaporated. The crude product was purified onreverse phase ISCO using 80 g C-18 column eluting with 0-95%acetonitrile in water (0.05% formic acid) to provide fractions with thedesired product, which were lyophilized to provide compound 53 as paleyellow paste (0.8 g). LCMS ESI: calculated for C₁₃H₂₆N₂O₃=259.3 (M+H⁺),found 159.1 (M-Boc+H⁺). δ 6.65 (s, 2H), 4.30 (d, J=9.4 Hz, 2H),3.75-3.67 (m, 2H), 3.60 (s, 2H), 2.84 (t, J=12.7 Hz, 2H), 2.00 (s, 3H),1.75 (dddd, J=14.1, 10.7, 5.3, 3.6 Hz, 1H), 1.44-1.24 (m, 9H), 0.91 (t,J=6.8 Hz, 3H).

Compound 54. Compound 53 (0.8 g, 3.10 mmol) was treated with TFA (4.7mL, 61.9 mmol). LCMS after 30 min shows completion of reaction. TFA wasevaporated. The residue was dried in a lyophilizer overnight to givecompound 54 (322 mg, 65.7% yield) as pale yellow paste. LCMS ESI:calculated for C₈H₁₅N20=159.2 (M+H⁺), found 159.1 (M+H⁺). δ 8.08 (s,2H), 6.71 (s, 1H), 3.49 (s, 1H), 3.38-3.32 (m, 2H), 3.30 (s, 1H), 3.18(d, J=9.7 Hz, 1H), 2.04 (s, 3H), 1.96-1.83 (m, 2H), 1.67 (ddq, J=36.7,14.4, 7.5, 7.0 Hz, 1H), 1.40 (dq, J=15.6, 7.7 Hz, 1H), 0.94 (t, J=7.3Hz, 3H).

Compound 54 can be used to make compounds such as 846 and 847, generallyfollowing the Procedure 3 and FIGS. 3A-3B, using compound 16.

Procedure 6—Compounds per FIGS. 6a -6B

This procedure and companion FIGS. 6A-6B illustrate another method ofmaking compounds disclosed herein, using compound 857 as an exemplar.

Compound 55. To a suspension of compound 30 (1.50 g, 7.27 mmol) in DMF(25 mL) was added NBS (1.29 g, 7.27 mmol) in three portions (colorchanged from red to light yellow). After 10 min stirring at RT, LCMSindicated that the reaction was complete. The mixture was poured onto asmall portion of ice and stirred for 16 h. The resulting solid wascollected by filtration and dried in high vacuum to yield compound 55(1.96 g, 6.9 mmol, 95% yield).

Compound 57. A suspension of 4-fluoro-2-methoxybenzaldehyde 56 (1.5 g,9.73 mmol), methyl 4-hydroxybenzoate 32 (1.777 g, 11.68 mmol) and K₂CO₃(2.69 g, 19.46 mmol) in DMF (19.46 ml) was stirred at 105° C. for threedays, LCMS indicated that reaction was complete. It was quenched withwater. The resulting precipitate of a creamy colored solid was collectedby filtration and dried in vacuo to yield compound 57 (2.6 g, 9.08 mmol,93% yield). LCMS ESI: calculated for C₁₆H₁₅O₅=287.1 (M+H⁺), found 287.1(M+H⁺-18).

Compound 58. To a stirred suspension of compound 57 (2.6 g, 9.08 mmol)in MeOH (25 mL) was added NaBH₄ (0.344 g, 9.08 mmol) in portions. Thereaction mixture turned clear. After 1 h, the reaction was complete. Thereaction mixture was concentrated to half-volume. Water was added,followed by stirring for 30 min. The resulting solid was collected byfiltration and air dried to yield compound 58 (2.50 g, 8.67 mmol, 95%yield) as a white solid, which was pure enough for use in the next step.LCMS ESI: calculated for C16H1705=289.1 (M+H⁺), found 271.1 (M+H⁺-18).

Compound 59. Compound 58 (730 mg, 2.53 mmol) in THF (10 mL) was treated,with stirring, with SOCl₂ (0.924 mL, 12.66 mmol). Stirring was continuedat RT for 16 h. Concentration on a rotary evaporator yielded compound59. The extra SOCl₂ was removed azetropically with DCM (3×). Theresulting solid was pure enough to carry over to next step withoutfurther purification. ¹H NMR (400 MHz, DMSO-d6) δ 8.07-7.83 (m, 2H),7.46 (d, J=8.1 Hz, 1H), 7.16-7.01 (m, 2H), 6.87 (d, J=2.2 Hz, 1H), 6.66(dd, J=8.3, 2.3 Hz, 1H), 4.73 (s, 2H), 3.84 (d, J=5.3 Hz, 6H).

Compound 60. To a stirred mixture of compound 55 (300 mg, 1.052 mmol) inDMF (1 mL) was added compound 59 (323 mg, 1.052 mmol). After stirring atRT for 3 h, no starting material 59 was detected. The reaction mixturewas quenched with brine, extracted with DCM (3×20 mL). The combinedorganic extracts were dried with Na₂SO₄ and filtered. The filtrate wasconcentrated. The crude mixture was purified by ISCO silica column (40g), eluting with EtOAc/hexanes=0-100%. The desired fractions wereconcentrated to yield compound 60 (166 mg, 0.299 mmol, 28.4% yield).LCMS ESI: calculated for C₂₂H₂₈BrN₆O₄=555.1, 557.1 (M+H⁺), found 555.2,557.2 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J=8.8 Hz, 2H),7.55-7.55 (m, 1H), 7.12-6.98 (m, 2H), 6.87 (d, J=2.2 Hz, 1H), 6.76 (t,J=5.4 Hz, 1H), 6.71 (d, J=8.1 Hz, 1H), 6.62 (dd, J=8.3, 2.3 Hz, 1H),5.97 (s, 2H), 5.62 (s, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.50-3.39 (m,2H), 1.59-1.46 (m, 2H), 1.31-1.21 (m, 2H), 0.87 (t, J=7.4 Hz, 3H).

Compound 61. To a stirred mixture of compound 60 (160 mg, 0.288 mmol) inTHF (10 mL) was added LiAlH₄ (2.5M in THF) (10.93 mg, 0.288 mmol) undernitrogen at RT. After 10 mins, LCMS indicated that reaction wascomplete. The reaction mixture was quenched with Rochelle salt solutioncarefully, and stirred at RT for 18 h. The organic layer was separatedand the aqueous layer was further extracted with ethyl acetate (2×50mL). The combined organic extracts were dried over Na₂SO₄ and filtered.The filtrate was concentrated to yield compound 61 (140 mg, 0.265 mmol,92.2% yield). LCMS ESI: calculated for C₂₄H₂₈BrN₆O₃=527.1, 529.1 (M+H⁺),found 527.2, 529.2 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.6Hz, 2H), 6.96 (d, J=8.6 Hz, 2H), 6.77-6.65 (m, 3H), 6.43 (dd, J=8.3, 2.3Hz, 1H), 5.95 (s, 2H), 5.57 (s, 2H), 5.15 (t, J=5.6 Hz, 1H), 4.47 (d,J=5.7 Hz, 2H), 3.76 (s, 3H), 3.45 (br d, J=5.7 Hz, 2H), 1.53 (t, J=7.2Hz, 2H), 1.31-1.20 (m, 2H), 0.88 (t, J=7.4 Hz, 3H).

Compound 62. A suspension of compound 61 (165 mg, 0.313 mmol) in MeOH(10 mL) and ethyl acetate (3 mL) was purged with N2. Pd—C 5% (40 mg,0.019 mmol) was added, purged with H2, and stirred under H2 balloon for18 h. The catalyst was then removed by filtration through a CELITE® pad.The filtrate was concentrated to yield compound 62 (157 mg, 0.350 mmol,112% yield). LCMS ESI: calculated for C₂₄H₂₉N₆O₃=449.2 (M+H⁺), found449.3 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 8.13 (br d, J=4.0 Hz, 1H),7.75 (s, 1H), 7.61 (br s, 2H), 7.33 (d, J=8.6 Hz, 2H), 6.97 (d, J=8.6Hz, 2H), 6.83 (d, J=8.6 Hz, 1H), 6.74 (d, J=2.2 Hz, 1H), 6.42 (dd,J=8.3, 2.3 Hz, 1H), 5.68 (s, 2H), 5.26-5.03 (m, 1H), 4.48 (d, J=5.3 Hz,2H), 3.73 (s, 3H), 3.58 (br d, J=6.2 Hz, 2H), 1.59 (br t, J=7.3 Hz, 2H),1.35-1.18 (m, 2H), 0.90 (t, J=7.4 Hz, 3H).

Compound 857. A suspension of compound 62 (100 mg, 0.223 mmol) in THF (4mL) was treated with SOCl₂ (0.081 mL, 1.115 mmol), followed by stirringat RT for 1 h. The reaction mixture was then concentrated on a rotaryevaporator. The excess SOCl₂ was azeotropically removed with DCM (3×),the resulting solid was pure enough for use in the next step. LCMS ESI:calculated for C₂₄H₂₈ClN₆O₂=467.2 (M+H⁺), found 467.2 (M+H⁺). Theresulting solid (20 mg, 0.043 mmol) was dissolved in DMF (0.5 mL).1-Amino-2-methylpropan-2-ol (19.09 mg, 0.214 mmol) was added to thereaction mixture and the solution was stirred at RT for 16 hrs. Thereaction mixture was syringe-filtered and the crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 200 mm×19 mm, 5-m particles; Mobile Phase A: 5:95acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: a 0-minutehold at 19% B, 19-59% B over 20 minutes, then a 0-minute hold at 100% B;Flow Rate: 20 mL/min; Column Temperature: 25 C. Fraction collection wastriggered by MS and UV signals. Fractions containing the desired productwere combined and dried via centrifugal evaporation to yield compound857 as a TFA salt (6.9 mg, 10.63 μmol, 24.81% yield).

This Procedure 6 and prior Procedure 4 can be used to make similarcompounds. We have observed that alkylation of 3-bromo compound 55yields a higher proportion of the 1H regioisomer (2H regioisomer notshown in FIG. 6A) than the alkylation of counterpart compound 36 inProcedure 4. Thus, where the 1H regioisomer is the more desired one,this Procedure 6 may be the preferable one, even though it entails theextra bromination and de-bromination steps. Table B below lists somecompounds that were made by either Procedure 4 or 6. Also, 3-bromocompound 55 can be an intermediate in other synthetic pathways, as shownin Procedures hereinbelow.

Procedure 7—Compounds Per FIG. 7

This Procedure and companion FIG. 7 illustrate another method for makingcompounds disclosed herein, using compound 864 as an exemplar.

Compound 64. A solution of compound 16 (48.5 mg, 0.125 mmol) in THF (1.8mL) was cooled to 0° C. and treated with LiAlH₄ (1M in THF, 219 μL,0.219 mmol). After 10 min, an additional portion of LiAlH₄ (1M in THF,200 μL, 0.200 mmol) was added and the reaction was stirred for another20 min at 0° C. The reaction mixture was quenched with MeOH andRochelle's salt (sat. aq. solution) and stirred at RT for 30 min. Themixture was extracted with EtOAc (3×). The combined organic layers werewashed with H₂O, dried over Na₂SO₄, filtered and concentrated in vacuoto give compound 64 (38 mg, 84%)). ¹H NMR (400 MHz, DMSO-d6) δ11.60-11.12 (m, 2H), 7.85-7.83 (m, 1H), 6.97 (s, 1H), 6.77 (d, J=8.0 Hz,1H), 6.59 (d, J=7.7 Hz, 1H), 5.66 (s, 2H), 5.15 (t, J=5.7 Hz, 1H), 4.44(d, J=5.6 Hz, 2H), 3.79 (s, 3H), 3.74 (s, 3H). LC/MS conditions B: LCRT: 0.61 min. LCMS (M+H)=360.1.

Compound 65. A solution of compound 64 (402 mg, 1.12 mmol) in DCM (15mL) was treated with thionyl chloride (1.6 mL, 22 mmol). After 15 min,the reaction mixture concentrated in vacuo. The residue was redissolvedin DCM and concentrated in vacuo again to yield compound 65 (422 mg,100%). ¹H NMR (400 MHz, DMSO-d6) δ 11.25-11.02 (m, 1H), 7.89-7.88 (m,1H), 7.11 (d, J=1.5 Hz, 1H), 6.92 (dd, J=7.8, 1.5 Hz, 1H), 6.62 (d,J=7.7 Hz, 1H), 5.69 (s, 2H), 4.72 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H).One proton was not visible, likely due to overlap with water peak orproton exchange. LC/MS conditions B: LC RT: 0.80 min. LCMS (M+H)=378.0.

Compound 66. A solution of cyclobutanamine 5a (0.84 mL, 9.9 mmol) in THF(11 mL) was treated a solution of compound 65 (249 mg, 0.659 mmol) inTHF (8 mL). The reaction mixture was stirred for 16 h at 60° C. and thenconcentrated in vacuo. The residue was dissolved in DCM/MeOH, absorbedonto CELITE® and purified via column chromatography (50 g C18 goldcolumn; Mobile Phase A: 10:90 methanol:water with 0.1% trifluoroaceticacid; Mobile Phase B: 90:10 methanol:water with 0.1% trifluoroaceticacid; Flow Rate: 40 mL/min) to give compound 66 as its TFA salt (168 mg,48% yield). ¹H NMR (400 MHz, DMSO-d6) δ 11.55 (br s, 1H), 11.18-11.12(m, 1H), 9.04-8.93 (m, 2H), 7.91-7.89 (m, 1H), 7.19 (d, J=1.4 Hz, 1H),6.95 (dd, J=7.7, 1.3 Hz, 1H), 6.68 (d, J=7.7 Hz, 1H), 5.71 (s, 2H),4.02-3.97 (m, 2H), 3.85 (s, 3H), 3.76 (s, 3H), 3.72-3.63 (m, 1H),2.20-2.10 (m, 4H), 1.85-1.72 (m, 2H). LC/MS conditions B: LC RT: 0.63min. LCMS (M+H)=413.3.

Compound 864. A solution of compound 66 (15 mg, 0.036 mmol) in DMF (1mL) was treated with BOP (161 mg, 0.364 mmol), 2-ethoxyethan-1-amine 6a(32.4 mg, 0.364 mmol) and DBU (73 μL, 0.49 mmol) and stirred at RT for18 h. The reaction mixture was concentrated under a stream of nitrogen.A solution of the crude product 67 in dioxane (0.5 mL) was treated withNaOH (10 M aq soln, 0.05 mL, 0.5 mmol) and heated to 75° C. After 1 h,the reaction mixture was neutralized with HOAc (0.03 mL, 0.5 mmol) andconcentrated under a stream of nitrogen. The residue was dissolved inDMF, filtered through a PTFE frit, and the crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Gradient: a 0-minute hold at 3% B,3-43% B over 25 minutes, then a 0-minute hold at 100% B; Flow Rate: 20mL/min; Column Temperature: 25° C. Fraction collection was triggered byMS signals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give compound 864 (7.3 mg, 48%yield). LC/MS conditions C. LC RT: 0.8 min. LCMS (M+H)=426.17.

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 8—Compounds Per FIG. 8

This Procedure and companion FIG. 8 illustrate another method for makingcompounds disclosed herein, using compound 866 as an exemplar.

Compound 70. A solution of compound 68 (307 mg, 0.922 mmol) inacetonitrile (0.9 mL) was treated with compound 69 (321 mg, 1.01 mmol)and stirred at RT for 72 h. The reaction mixture was then heated to 60°C. for 4 h, recooled to RT and filtered. The collected solid was rinsedwith acetonitrile and dried in vacuo. The filtrate was concentrated invacuo and the residue purified by column chromatography (12 g SiO₂, 0 to30% EtOAc-hexane, gradient elution) to give a white solid, which wascombined with the solid isolated by filtration to give compound 70 (392mg, 74%). LC/MS conditions B: LC RT: 1.21 min. LCMS (M+H)=576.9.

Compound 71. A solution of compound 70 (144 mg, 0.251 mmol) in MeOH (1.2mL) was treated with NaOMe (81 mg, 1.5 mmol) and stirred at RT. After 96h, the reaction mixture was diluted with H₂O and extracted with EtOAc(3×). The combined organic layers were dried over Na₂SO₄, filtered andconcentrated in vacuo to give the compound 71 (108 mg, 100% yield). ¹HNMR (400 MHz, DMSO-d6) δ 11.62-11.59 (m, 1H), 10.85-10.82 (m, 1H), 7.91(s, 1H), 7.51 (d, J=1.4 Hz, 1H), 7.47 (dd, J=7.9, 1.4 Hz, 1H), 6.69 (d,J=7.9 Hz, 1H), 5.75 (s, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 1.50 (s, 9H).LC/MS conditions B: LC RT: 0.89 min. LCMS (M+H)=430.6.

Compound 72. A solution of compound 71 (294 mg, 0.685 mmol) in DMSO (3.4mL) was treated with (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine(71a) hydrochloride (322 mg, 0.822 mmol), BOP (364 mg, 0.822 mmol) andDBU (516 μL, 3.43 mmol) and stirred at RT. After 3 h, the reactionmixture diluted with EtOAc and washed with H₂O. The organic layerconcentrated in vacuo and the residue was purified via columnchromatography (24 g SiO₂; 0 to 25% EtOAc-DCM, gradient elution) to givecompound 72 (227.8 mg, 43%). ¹H NMR (400 MHz, DMSO-d6) δ 9.00-8.93 (m,1H), 7.92 (s, 1H), 7.53 (dd, J=8.0, 1.4 Hz, 2H), 7.46 (s, 2H), 7.44 (d,J=1.3 Hz, 1H), 7.42-7.37 (m, 1H), 7.37-7.31 (m, 3H), 7.24-7.20 (m, 2H),7.20-7.16 (m, 1H), 6.28 (d, J=7.9 Hz, 1H), 6.20 (d, J=8.5 Hz, 1H),5.83-5.72 (m, 2H), 4.60-4.46 (m, 1H), 3.85 (s, 3H), 3.76 (s, 3H),3.55-3.43 (m, 2H), 1.83-1.73 (m, 1H), 1.73-1.65 (m, 1H), 1.46 (br d,J=13.6 Hz, 2H), 1.41 (s, 9H), 1.15-1.04 (m, 2H), 0.91 (s, 9H), 0.74 (t,J=7.3 Hz, 3H). LC/MS conditions B: LC RT: 1.07 min. LCMS (M+H)=767.4.

Compound 73. A solution of methyl ester 72 (227 mg, 0.297 mmol) in THF(4.2 mL) was cooled to 0° C. and treated with LiAlH₄ (1M in THF, 327 μL,0.327 mmol). After 10 min, additional LiAlH₄ (1M in THF, 150 μL, 0.150mmol) added. After 30 min, the reaction was quenched with MeOH andRochelle's salt (sat aq soln) and stirred at RT for 30 minutes. Themixture was extracted with EtOAc (3×). The combined organic layers werewashed with H₂O, and concentrated in vacuo. The residue was purified viacolumn chromatography (12 g SiO₂; 0 to 100% EtOAc-hexane, gradientelution) to give compound 73 (91.5 mg, 42%). LC/MS conditions B: LC RT:1.02 min. LCMS (M+H)=739.7.

Compound 74. A solution of compound 73 (91.5 mg, 0.124 mmol) in THF (1.2mL) was treated with thionyl chloride (45 μL, 0.61 mmol) and stirred atRT. After 15 min, the reaction mixture concentrated in vacuo, theresidue was redissolved in DCM and concentrated in vacuo to providecompound 74 (94 mg, 100%). LC/MS conditions B: LC RT: 1.09 min. LCMS(M+H)=757.3.

Compound 75. A solution of compound 74 (20 mg, 0.027 mmol) in DMF (0.5mL) was treated with DIEA (70 μL, 0.40 mmol) and3-(tert-butyl)cyclobutan-1-amine 74a (34.1 mg, 0.268 mmol) and heated to70° C. for 1 h. The reaction mixture was cooled to RT and concentratedin vacuo. The residue was dissolved in EtOAc and washed with sat. aqNaHCO₃ soln. The organic layer was concentrated in vacuo to give affordcompound 75 (22 mg, 97%), which was used without further purification.LC/MS conditions B: LC RT: 0.95 min. LCMS (M+H)=848.6.

Compound 866. A solution of compound 75 (25 mg, 0.029 mmol) in MeOH (0.6mL) was treated with HCl (37% aq soln, 0.3 mL, 3.7 mmol) and stirred atRT for 6 h. The reaction mixture was neutralized with NaHCO₃ andextracted with DCM (3×). The combined organic layers were concentratedin vacuo. The residue was redissolved in DMF and filtered through a PTFEfilter. The crude product was purified via preparative HPLC/MSconditions A-1: Fraction collection was triggered by MS signals.Fractions containing the desired product were combined and dried viacentrifugal evaporation to give compound 866 (3.3 mg, 22% yield). LC/MSconditions C: LC RT=1.23 min. LCMS (M+H)=510.23.

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 9—Compounds Per FIG. 9

This Procedure and companion FIG. 9 illustrate another method for makingcompounds disclosed herein, using compounds 853 and 870 as exemplars.

Compound 76. A solution of compound 55 (400 mg, 1.40 mmol) and cesiumcarbonate (914 mg, 2.8 mmol) in DMF/acetonitrile (5 mL/5 mL) was cooledin an ice bath. A solution of methyl 4-(bromomethyl)-3-fluorobenzoate55a (277 mg, 1.12 mmol) in acetonitrile (5 mL) was added, and thereaction stirred at 0° C. for 30 min, then at RT for a further hour. Theacetonitrile was evaporated from the reaction mixture and purified usingreverse-phase flash chromatography (100 g column, 10 to 60% MeCN inwater containing 0.05% TFA) to give compound 76 (482 mg, 1.07 mmol, 35%purity (contaminated with the N2-regioisomer, 27% yield) as a solid.LC-MS (ES, m/z): [M+H]+=451.1, 453.2.

Compound 77. Compound 76 (470 mg, 1.04 mmol, 35% purity (contaminatedwith the N2-regioisomer) was dissolved in ethanol (50 mL). 10% Pd/C (20mg) was added, and the reaction evacuated and purged with hydrogen sixtimes, then stirred under a hydrogen atmosphere for 90 min. The reactionmixture was filtered through CELITE®, washed with EtOH (100 mL), andevaporated to dryness, giving compound 77 (370 mg, 0.99 mmol, 35% purity(contaminated with the N2-regioisomer), 95% yield) as a solid. LC-MS(ES, m/z): [M+H]⁺=373.3.

Compound 78. A stirred suspension of compound 77 (370 mg, 0.99 mmol, 35%purity contaminated with the N2-regioisomer) in THF (100 mL) was cooledin an ice bath. LiAlH₄ (754 mg, 1.98 mmol) was added portion wise over 5min, and the reaction was stirred at 0° C. for 20 minutes. NaOH (1N, 50mL) was added, and the reaction stirred for 10 min at RT, transferred toa separatory funnel and extracted with EtOAc (3×40 mL). The combinedorganic phases were washed with brine (3×30 mL), dried (MgSO₄), filteredand concentrated. Flash chromatography (40 g column, 0 to 25% MeOH inDCM) yielded compound 78 (74 mg, 0.215 mmol, 21.63% yield) as a solid.LC-MS (ES, m/z): [M+H]⁺=345.3.

Compound 853. A 20 mL scintillation vial was charged with compound 78(25 mg, 0.073 mmol) and THF (4 mL). 3 drops of thionyl chloride wereadded, and the reaction mixture was stirred for 1 hour. The reactionmixture was evaporated to dryness and the residue was dissolved inacetonitrile (5 mL) and evaporated twice. The residue was dissolved inDMF (1.5 mL). Cyclobutanamine (20.7 mg, 0.29 mmol) was added, and thereaction mixture stirred at RT overnight. The reaction mixture wasfiltered and purified via preparative HPLC/MS conditions A-1: Fractioncollection was triggered by MS and UV signals. Fractions containing thedesired product were combined and dried via centrifugal evaporation togive compound 853 (19.5 mg, 0.03 mmol, 42% yield).

Compound 870. A 20 mL scintillation vial was charged with compound 78(50 mg, 0.15 mmol) and THF (4 mL). 3 Drops of thionyl chloride wereadded, and the reaction mixture stirred for 1 h. The reaction mixturewas evaporated to dryness. The residue was dissolved in acetonitrile (5mL) and evaporated twice. The residue was dissolved in DMF (2 mL).2-(piperazin-1-yl)ethan-1-ol (38 mg, 0.29 mmol) was added, followed byDIPEA (0.10 mL, 0.58 mmol), and the reaction mixture stirred at 50° C.for 1 h. After cooling, the reaction mixture was filtered and purifiedvia preparative HPLC/MS conditions A-1: Fraction collection wastriggered by MS and UV signals. Fractions containing the desired productwere combined and dried via centrifugal evaporation, giving compound 870(41 mg, 0.06 mmol, 42% yield).

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 10—Compounds Per FIG. 10

This Procedure and companion FIG. 10 illustrate another method formaking compounds disclosed herein, using compound 873 as an exemplar.

Compound 80. A 40 mL scintillation vial was charged with compound 79(250 mg, 1.55 mmol), NBS (331 mg, 1.86 mmol), AIBN (50.9 mg, 0.31 mmol)and CCl₄ (5 mL). The reaction mixture was stirred at 70° C. for 2 h.After cooling, the reaction mixture was evaporated to dryness. Flashchromatography (24 g column, 0 to 40% EtOAc in hexanes) gave compound 80(189 mg, 0.787 mmol, 51% yield) as a solid. LC-MS (ES, m/z): No iondetected. ¹H NMR (400 MHz, CDCl₃) δ 7.33 (d, J=7.7 Hz, 1H), 6.88 (d,J=7.7 Hz, 1H), 6.84 (s, 1H), 4.53 (s, 2H), 3.92 (s, 3H), 3.74 (s, 2H).

Compound 81. To a stirred suspension of methyl(7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (6 g, 22.7mmol) in DMF (10 mL) and was added a solution of NBS (4.44 g, 25.0 mmol)in acetonitrile (20 mL). The reaction mixture was stirred at RT for 1 h.Water (50 mL) was added. The reaction mixture filtered. The residue waswashed with water (3×30 mL) and air dried overnight to yield compound 81(5.112 g, 14.9 mmol, 66% yield) as a solid. LC-MS (ES, m/z):[M+H]⁺=343.0, 345.0. ¹H NMR (400 MHz, DMSO-d6) δ 12.87 (br s, 1H), 9.80(s, 1H), 7.56 (br s, 1H), 3.62 (s, 3H), 3.54 (q, J=6.6 Hz, 2H), 1.62(quin, J=7.2 Hz, 2H), 1.40 (dq, J=14.8, 7.4 Hz, 2H), 0.94 (t, J=7.4 Hz,3H).

Compound 82. Compound 81 (1 g, 2.91 mmol) was dissolved in DMF (10 mL)and cooled in an ice bath. Cesium carbonate (1.899 g, 5.83 mmol) wasadded, followed by compound 80 (0.560 g, 2.33 mmol). The reactionmixture was stirred at RT for 5 h and then poured into saturated NaHCO₃solution (50 mL) and extracted with EtOAc (3×40 mL). The combinedorganic phases were washed with brine (4×40 mL), dried (MgSO₀₄),filtered and concentrated. Flash chromatography (40 g column, 0 to 85%EtOAc in hexane) gave compound 82 (219 mg, 0.44 mmol, 15% yield) as asolid. LC-MS (ES, m/z): [M+H]⁺ 502.2, 504.2. ¹H NMR (400 MHz, CDCl₃) δ7.25-7.19 (m, 1H), 6.98 (s, 1H), 6.97-6.91 (m, 1H), 5.70 (s, 2H), 5.28(s, 1H), 3.98 (s, 3H), 3.84 (s, 3H), 3.78-3.60 (m, 4H), 1.72-1.41 (m,2H), 1.41-1.20 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).

Compound 83. Compound 82 (219 mg, 0.44 mmol) was dissolved in EtOH (10mL) and 10% Pd/C (20 mg) was added. The reaction vessel was evacuatedand purged with H2 six times, then stirred under an H2 atmosphere for 2h. The reaction mixture was filtered and evaporated to dryness, givingcompound 83 (188 mg, 0.44 mmol, 100% yield) as a solid. LC-MS (ES, m/z):[M+H]⁺ 424.4. ¹H NMR (400 MHz, DMSO-d6) δ 11.71 (br s, 1H), 8.67 (br s,1H), 8.04 (s, 1H), 7.05 (s, 1H), 6.89 (s, 2H), 5.77 (s, 2H), 4.01 (s,2H), 3.83 (s, 3H), 3.76 (s, 3H), 3.69-3.58 (m, 2H), 1.72-1.48 (m, 2H),1.41-1.17 (m, 2H), 0.90 (t, J=7.4 Hz, 3H).

Compound 873. Compound 83 (25 mg, 0.059 mmol) was dissolved in dioxane(2 mL); sodium hydroxide (0.354 mL, 1.77 mmol) was added. The reactionmixture was stirred at 80° C. for 3 h. After cooling, the reaction wasneutralized with 5N HCl, then evaporated to dryness. The residue wasdissolved in DMF (2 mL), filtered and purified via preparative HPLC/MSconditions A-2: Fraction collection was triggered by MS signals.Fractions containing the desired product were combined and dried viacentrifugal evaporation to give compound 873 (6.2 mg, 0.013 mmol, 22%).

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 11—Compounds Per FIG. 11

This Procedure and companion FIG. 11 illustrate another method formaking compounds disclosed herein, using compound 855 as an exemplar.

Compound 85. To a solution of methyl 5-methoxy-6-methylnicotinate 84(300 mg, 1656 μmol) and NBS (413 mg, 2318 μmol) in CCl₄ (3 mL), AIBN(54.4 mg, 331 μmol) was added. The reaction mixture was stirred at 80°C. for 30 min. LCMS analysis showed the reaction was complete. Thereaction mixture was cooled down and diluted with DCM (2 mL). Theresulting solution was purified on a 40 g RediSepRf Gold Silica column.Mobile phase A: hexanes; Mobile phase B: Ethyl acetate; Gradient: 1 minat 0% B, 15 min at 0-50% B; Flow rate: 40 mL/min; Column Temperature:25° C. Fraction collection was triggered by UV signals at 254 nm. Thefractions containing expected product were combined, concentrated anddried under high vacuum to give compound 85 (215 mg, 50%). LC/MSconditions E: LC RT: 1.57 min. [M+H]+/Z=260.0

Compound 86. To a solution of compound 30 (427 mg, 2070 μmol) in DMF (2mL), Cs₂CO (701 mg, 2152 μmol) was added. The reaction mixture wasstirred at RT for 10 min. A solution of compound 85 (215 mg, 0.827 mmol)in acetonitrile (2 mL) was added to the reaction mixture slowly. Thereaction mixture was stirred at RT for 30 min. LCMS analysis showed 3peaks corresponding product m/z. The reaction mixture was neutralizedwith acetic acid (0.3 mL), diluted with water and purified bypreparative HPLC/MS conditions A-3: Fraction collection was triggered byUV signals. The fractions corresponding to the three peaks werefreeze-dried separately. The NMR analysis confirmed that compoundcorresponding to the latest peak was the desired compound 86 (76 mg,11.9%). LC/MS conditions E: LC-RT: 1.89 min. [M+H]+/Z=386.2. ¹H NMR (500MHz, DMSO-d6) δ 8.57 (t, J=5.7 Hz, 1H), 8.50 (d, J=1.6 Hz, 1H), 7.88 (d,J=1.6 Hz, 1H), 7.83 (s, 1H), 7.76 (s, 1H), 5.95 (s, 2H), 4.00 (s, 3H),3.89 (s, 3H), 3.55 (q, J=6.8 Hz, 2H), 1.59-1.48 (m, 2H), 1.30-1.14 (m,2H), 0.86 (t, J=7.4 Hz, 3H).

Compound 87. To a solution of compound 86 (38 mg, 0.099 mmol) in THF (1mL), LiAlH₄ in THF (0.079 mL, 0.197 mmol) was added. The reactionmixture was stirred at RT for 10 min. LCMS analysis showed the reactionwas complete. The reaction mixture was neutralized with acetic acid (0.2mL), diluted with water (3 mL) and purified on HPLC/MS conditions A-3:Fraction collection was triggered by UV signals. The fractionscontaining expected product were combined and freeze-dried to givecompound 87 (28 mg, 79%). LC/MS conditions E: LC-RT: 1.26 min.[M+H]+/Z=357.2.

Compound 855. To a solution of compound 87 (28 mg, 0.078 mmol) in DCM (1mL), SOCl₂ (0.144 mL, 1.972 mmol) was added. The reaction mixture wasstirred at RT overnight. The reaction mixture was concentrated andco-evaporated with DCM (3×20 mL) to form crudeN7-butyl-1-((5-(chloromethyl)-3-methoxypyridin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine(41 mg), which was used for next step immediately without purification.

To a solution of the preceding compound (41 mg, 0.109 mmol, crude) inDMF (1 mL), cyclobutanamine (388 mg, 5.45 mmol) was added. The reactionmixture was stirred at RT for 3 h. LCMS analysis showed the reaction wascomplete. The reaction mixture was freeze-dried with acetonitrile andwater. The crude material was purified via preparative HPLC with thefollowing conditions: Column: XBridge C18, 200 mm×19 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate;Gradient: a 0-minute hold at 8% B, 8-48% B over 20 minutes, then a0-minute hold at 100% B; Flow rate: 20 mL/min; Column Temperature: 25 C.Fraction collection was triggered by MS and UV signals. Fractionscontaining the desired product were combined and dried via centrifugalevaporation to give 6.6 mg (14.8%) of compound 855 (6.6 mg, 14.8%).LC/MS conditions: Column: Waters Aquity BEH C18, 2.1 mm×50 mm, 1.7 μmparticles; Mobile Phase A: 5:95 acetonitrile: water with 10 mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile: water with 10 mM ammoniumacetate; Temperature: 50° C.; Gradient: 0% B to 100% B over 3 min, thena 0.50 min hold at 100% B; Flow rate: 1 mL/min; Detection: MS and UV(220 nm). LC RT=1.36 min. (M+H)=411.1

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 12—Compounds Per FIG. 12

This Procedure and companion FIG. 12 illustrate another method formaking compounds disclosed herein, using compound 867 as an exemplar.

Compound 90. To a solution of compound 89 (300 mg, 0.722 mmol) dissolvedin DCM (3 mL) at −78° C. was added ethyl magnesium bromide (0.633 mL,2.165 mmol). 2-methyltetrahydrofuran was added slowly to the reactionmixture and stirred at −78° C. for 1 hr and then 0° C. for 1 h. Thereaction mixture was quenched with saturated ammonium chloride solution(1 mL) and brought to RT, then diluted with DCM (10 ml), washed withwater (5 mL), then followed by brine solution (5 ml). The organic layerwas dried over sodium sulphate, filtered, concentrated, purified onsilica gel with ethyl acetate/hexane. The product containing fractionswere concentrated to afford compound 90 as thick oil (204 mg, 63.4%yield). ¹H NMR (400 MHz, Chloroform-d) δ 7.58 (m, 4H), 7.42-7.26 (m,6H), 3.75-3.57 (m, 2H), 3.31 (d, J=7.8 Hz, 1H), 2.93 (d, J=7.5 Hz, 1H),1.75 (m, 1H), 1.68-1.47 (m, 3H), 1.06 (s, 9H), 0.98 (s, 9H), 0.87 (t,J=7.4 Hz, 3H). LCMS: M/Z=446.2.

Compound 91. Hydrochloric acid (194 μL, 0.774 mmol, 4 M in dioxane) wasadded to a solution of compound 90 (203 mg, 0.455 mmol) in diethyl ether(4554 μL) and MeOH (55.3 μL, 1.366 mmol). The mixture was stirred at RTfor 1 h. The solution was concentrated to a thick oil. 20 mL of hexanewas then added to the oil and the flask was kept in the freezer at −20°C. for 1 h. The white solid was collected and washed with cold hexanes,then dried under vacuum to give compound 91 (116 mg, 0.307 mmol, 67.4%yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.85 (s, 3H), 7.63(ddd, J=7.7, 3.0, 1.7 Hz, 4H), 7.56-7.40 (m, 6H), 3.92-3.65 (m, 2H),3.18 (p, J=6.3 Hz, 1H), 1.86 (dq, J=13.0, 6.5 Hz, 1H), 1.76 (dq, J=13.4,6.4 Hz, 1H), 1.64-1.47 (m, 2H), 1.01 (s, 9H), 0.89 (t, J=7.5 Hz, 3H).LCMS: M/Z=342.2.

Compound 867. A solution of compound 88 (30 mg, 0.068 mmol) in DMSO(0.75 mL) was treated with compound 91 (51.3 mg, 0.136 mmol), DBU (51.1μL, 0.339 mmol) followed by BOP (60.0 mg, 0.136 mmol) and heated at 70°C. for 2 h to give compound 92, which was not isolated. 10M aq. solutionof sodium hydroxide (250 μl, 2.500 mmol) was added to the reactionmixture of compound 92 and stirred at 75° C. for overnight. The reactionmixture concentrated in vacuo and the residue was treated with MeOH (0.3mL) and HCl (37% aq. solution, 0.3 mL, 3.7 mmol), stirred at RT for 30min and concentrated. The crude product was dissolved in DMF, filteredthrough a PTFE frit, and purified via preparative HPLC conditions A-1.Fraction collection was triggered by MS signals. Fractions containingthe desired product were combined and dried via centrifugal evaporationto yield compound 867 as white solid (13 mg, 40% yield).

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 13—Compounds Per FIG. 13

This Procedure and companion FIG. 13 illustrate another method formaking compounds disclosed herein, using compound 851 as an exemplar.

Methyl 6-(hydroxymethyl)nicotinate 94 was coupled with compound 55 andthen taken to compound 851, following the reaction conditions describedhereinabove for the various steps, mutatis mutandis.

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 14—Compounds Per FIG. 14

This Procedure and companion FIG. 14 illustrate another method formaking compounds disclosed herein, using compound 877 as an exemplar.

Compound 102. A solution of methyl 4-nitro-1H-pyrazole-5-carboxylate 100(1.00 g, 5.61 mmol) and K₂CO₃ (0.93 g, 6.73 mmol) in DMF (5.9 mL) wascooled to 0° C. and methyl 4-(bromomethyl)benzoate 101 (1.29 g, 5.61mmol) was added in portions. The reaction mixture was allowed to warm toRT and stirred for 4 h. The reaction mixture was concentrated in vacuo.The residue was dissolved EtOAc and washed with sat. NaHCO₃ and H₂O(2×). The organic layer was concentrated in vacuo. The residue wasdissolved in DCM and purified via column chromatography (80 g SiO₂, 0 to50% EtOAc-hexane gradient) to give compound 102 (0.37 g, 21%). ¹H NMR(400 MHz, CHLOROFORM-d) δ 8.10-8.08 (m, 1H), 8.06-8.01 (m, 2H), 7.32 (d,J=8.6 Hz, 2H), 5.55 (s, 2H), 3.93 (s, 3H), 3.92-3.92 (m, 3H). LC/MScondition B. LC RT: 0.95 min. LCMS (M+H)=320.1.

In the preceding reaction, some of the N2 regioisomer was produced,which was removed during the SiO₂ chromatography:

Compound 103. Ammonium formate (292 mg, 4.64 mmol) and zinc (189 mg, 2.9mmol) were added to a solution of compound 102 (370 mg, 1.16 mmol), inTHF (1.5 ml)/MeOH (1.5 ml) at RT. The reaction was stirred at RT for 3 hand additional portions of ammonium formate (100 mg, 1.59 mmol) and zinc(100 mg, 2.04 mmol) were added. After 30 min, the reaction mixturefiltered through a pad of CELITE™, and the filtrate was concentrated invacuo to afford a white solid. The solid was suspended in EtOAc, stirredfor 30 minutes and filtered. The organic filtrate was then concentratedin vacuo to give compound 103 (335 mg, 100%). ¹H NMR (400 MHz, DMSO-d6)δ 7.93-7.86 (m, 2H), 7.19-7.15 (m, 3H), 5.59 (s, 2H), 5.13 (s, 2H), 3.83(s, 3H), 3.73 (s, 3H). LC/MS condition B: LC RT: 0.78 min. LCMS(M+H)=290.1.

Compound 104. 1,3-bis(Methoxycarbonyl)-2-methyl-thiopseudourea 1 (271mg, 1.27 mmol) and acetic acid (0.994 mL, 17.37 mmol) were added to asolution of compound 103 (335 mg, 1.16 mmol) in MeOH (14 mL) at RT. Thereaction mixture was stirred at RT for 3 h and an additional portion ofHOAc (0.5 mL, 8.7 mmol) was added. After 16 h at RT another portion ofHOAc (0.3 mL, 5.2 mmol) was added. After 72 h, sodium methoxide solution(7.94 mL, 34.7 mmol, 25% in MeOH) was added to the reaction mixture.After stirring for 2 h, the reaction mixture was acidified with HOAc (3mL) and the resulting slurry was filtered. The resulting solid waswashed with H₂O and MeOH, and dried overnight to afford compound 104(383 mg, 93%). ¹H NMR (400 MHz, DMSO-d6) δ 11.52-11.22 (m, 1H),7.94-7.88 (m, 3H), 7.33 (d, J=8.3 Hz, 2H), 5.79 (s, 2H), 3.83 (s, 3H),3.74 (s, 3H). One proton not visible, likely due to proton exchange.LC/MS condition B: LC RT: 0.80 min. LCMS (M+H)=358.2.

Compound 105. (S)-1-((tert-Butyldiphenylsilyl)oxy)hexan-3-amine (71a)hydrochloride (840 mg, 2.14 mmol), BOP (711 mg, 1.61 mmol) and DBU (808μL, 5.36 mmol) were added sequentially to a RT solution of compound 104(383 mg, 1.07 mmol) in DMF (5.4 mL). The reaction mixture was stirredfor 16 h. At the end of the reaction, the mixture was diluted with EtOAcand washed with H₂O (2×). The organic layer was concentrated in vacuo.The residue was dissolved in DCM and purified via column chromatography(40 g SiO₂, 0 to 80% EtOAc-hexane gradient) to give provide compound 105(424 mg, 57%). ¹H NMR (400 MHz, DMSO-d6) δ 9.53-9.49 (m, 1H), 7.97 (s,1H), 7.66 (d, J=8.4 Hz, 2H), 7.64-7.59 (m, 1H), 7.54-7.50 (m, 2H),7.47-7.44 (m, 1H), 7.42-7.39 (m, 2H), 7.36-7.31 (m, 2H), 7.31 (br t,J=1.4 Hz, 1H), 7.19-7.12 (m, 2H), 6.94 (d, J=8.4 Hz, 2H), 6.32 (d, J=8.6Hz, 1H), 6.03-5.84 (m, 2H), 4.56 (td, J=8.4, 4.4 Hz, 1H), 3.72 (s, 3H),3.56 (s, 3H), 3.49-3.38 (m, 1H), 1.84-1.68 (m, 2H), 1.55-1.39 (m, 2H),1.36-1.21 (m, 1H), 1.18-1.07 (m, 1H), 0.91-0.86 (m, 9H), 0.74 (t, J=7.3Hz, 3H). LC/MS condition B: LC RT: 1.08 min. LCMS (M+H)=695.6.

Compound 106. A solution of compound 105 (424 mg, 0.610 mmol) in THF(8.7 mL) was cooled to −15° C. and LiAlH₄ (1M in THF, 1.1 mL, 1.1 mmol)was added. After 15 min an additional portion of LiAlH₄ (1M in THF, 0.3mL, 0.3 mmol) was added. After 10 min the reaction was quenched withMeOH and Rochelle's salt (sat. soln) and was stirred at RT for 30 min.The mixture was extracted with EtOAc (3×). The combined organic layerswere washed with H₂O, and concentrated in vacuo. The residue wasdissolved in DCM/MeOH, absorbed onto CELITE™ and purified via columnchromatography (24 g SiO₂; 0 to 100% EtOAc-hexane gradient elution) toafford compound 106 (181 mg, 44%). ¹H NMR (400 MHz, DMSO-d6) δ 9.47 (s,1H), 7.91 (s, 1H), 7.62 (ddt, J=5.7, 3.6, 1.8 Hz, 1H), 7.56 (dd, J=7.9,1.5 Hz, 2H), 7.48-7.43 (m, 3H), 7.39-7.32 (m, 3H), 7.24-7.20 (m, 2H),7.08 (d, J=8.1 Hz, 2H), 6.91 (d, J=8.1 Hz, 2H), 6.29 (d, J=8.6 Hz, 1H),5.78 (d, J=1.9 Hz, 2H), 5.06 (t, J=5.6 Hz, 1H), 4.63-4.54 (m, 1H), 4.32(d, J=5.4 Hz, 2H), 3.57 (s, 3H), 3.55-3.49 (m, 1H), 1.85-1.76 (m, 2H),1.55-1.40 (m, 2H), 1.20-1.02 (m, 2H), 0.91 (s, 9H), 0.78 (t, J=7.3 Hz,3H). LC/MS condition B: LC RT: 0.95 min. LCMS (M+H)=667.6.

Compound 107. Thionyl chloride (99 μL, 1.4 mmol) was added to a RTsolution of methyl compound 106 (181 mg, 0.224 mmol) in THF (2.7 mL).After stirring for 10 min, the reaction mixture was concentrated invacuo. The residue was redissolved in DCM and concentrated in vacuo togive compound 107 (172.2 mg, 93%). LC/MS condition B: LC RT: 1.11 min.LCMS (M+H)=685.6.

Compound 108. Compound 107 (23 mg, 0.034 mmol) was dissolved in DMF (0.7mL) at RT and was treated with DIEA (88 μL, 0.50 mmol) and3-methoxyazetidine (26a), as its hydrochloride (21 mg, 0.17 mmol). Afterstirring for 2 h at 70° C., the reaction mixture was concentrated invacuo to give crude compound 108, which was used without furtherpurification. LC/MS condition B: LC RT: 0.89 min. LCMS (M+H)=736.7.

Compound 877. A solution of compound 108 (25 mg, 0.034 mmol) in dioxane(0.7 mL) at RT was treated with NaOH (10M aq. soln, 0.1 mL, 1.0 mmol).The reaction was heated to 75° C. for 2 h, before the addition ofanother portion of NaOH (10M aq. soln, 0.15 mL, 1.5 mmol). After 4 hheating at 80° C., the reaction mixture was concentrated in vacuo. Theresidue was treated with MeOH (0.5 mL), HCl (37% aq. soln, 0.4 mL, 4.9mmol) was added, stirred at RT for 60 min, and concentrated. The crudeproduct was dissolved in DMF, filtered through a PTFE frit, and purifiedvia preparative LC/MS Condition A-2. Fraction collection was triggeredby MS signals. Fractions containing the desired product were combinedand dried via centrifugal evaporation to give a residue which wasfurther purified via preparative LC/MS Conditions A-1. Fractioncollection was triggered by MS signals. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation to givecompound 877, as its TFA salt (5.7 mg, 38%). LC/MS condition C: LC RT:1.0 min. LCMS (M+H)=440.2.

Compound 874. A solution of compound 106 (20 mg, 0.030 mmol) in dioxane(0.15 mL) at RT was treated with NaOH (10M aq. soln, 0.2 mL, 2.0 mmol)and the reaction was heated to 75° C. for 1 h, before the addition ofanother portion of NaOH (10M aq. soln, 0.2 mL, 2.0 mmol). After 3 hheating at 75° C., the reaction mixture was concentrated in vacuo. Theresidue was treated with MeOH (0.5 mL) and HCl (37% aq. soln, 0.4 mL,4.9 mmol), stirred at RT for 30 min and concentrated. The crude productwas dissolved in DMF, filtered through a PTFE frit, and purified viapreparative LC/MS conditions A-2. Fraction collection was triggered byMS signals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give compound 874. LC/MS conditionsC: LC RT: 1.07 min. LCMS (M+H)=371.2.

Additional compounds according to this disclosure can be made followingthis Procedure, mutatis mutandis.

Procedure 15—Compound 829

This procedure relates to the preparation of compounds 829 and itsregioisomer. These compounds were prepared by reacting compounds 36 and37 with MeMgCl Grignard reagent.

To a mixture of compounds 36 and 37 (60 mg, 0.134 mmol) in THF (1 mL)was added MeMgCl (0.171 mL, 0.513 mmol) at 0° C. After 1 hr, LCMS showedreaction was completed. The mixture was purified via preparative HPLC/MSwith the following conditions: Column: XBridge C18, 200 mm×19 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% TFA;Mobile Phase B: 95:5 acetonitrile: water with 0.1% TFA; Gradient: a0-minute hold at 10% B, 10-45% B over 20 minutes, then a 4-minute holdat 100% B; Flow Rate: 20 mL/min; Column Temperature: 25° C. Fractioncollection was triggered by MS signals. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation.

Properties

Analytical data for compounds of this disclosure are provided infollowing Table B, along with their human TLR7 (hTLR7) agonism data. Insome instances, the reported activity agonism value is the average ofplural assays. The association of a particular compound with aparticular Procedure is illustrative, and not exhaustive. A compoundcould be made by one of the other Procedures and, conversely, the sameProcedure could be used to make other compounds of this disclosure. TheRT (retention time) column refers to the retention time per LCMSprocedure B/C/D/E above, as applicable.

TABLE B hTLR7 Cpd. EC₅₀ LC/MS RT ¹H NMR No. Procedure (nM) [M + H]⁺(min) (500 MHz, DMSO-d₆) 800 1 58.3 409.5 1.05 δ 7.55 (s, 1H), 7.04 (s,1H), 6.78 (d, J = 7.7 Hz, 1H), 6.43 (dd, J = 21.5, 6.9 Hz, 2H), 5.61 (d,J = 11.9 Hz, 4H), 3.84 (s, 3H), 3.61 (s, 1H), 3.43- 3.37 (m, 1H), 3.18(t, J = 7.8 Hz, 1H), 2.55 (s, 1H), 2.09-2.02 (m, 2H), 1.91 (s, 1H),1.77- 1.69 (m, 2H), 1.67-1.59 (m, 1H), 1.58-1.43 (m, 3H), 1.20 (p, J =7.4 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H) 801 2 160 439.5 0.87 δ 8.22 (s,1H), 7.73 (s, 1H), 7.18 (s, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.91 (d, J =7.6 Hz, 1H), 5.71 (s, 2H), 4.13 (s, 2H), 3.92 (d, J = 9.8 Hz, 2H), 3.77(s, 1H), 3.64 (s, 1H), 3.57 (d, J = 6.6 Hz, 1H), 3.30 (t, J = 11.6 Hz,3H), 2.89 (s, 1H), 2.73 (s, 1H), 1.99 (d, J = 12.3 Hz, 2H), 1.63- 1.52(m, 4H), 1.28 (h, J = 7.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H) 802 4, 643.8 492.2 1.21 δ 7.55 (s, 1H), 7.36 (d, J = 8.5 Hz, 2H), 6.95 (d, J =8.5 Hz, 2H), 6.76 (d, J = 2.1 Hz, 1H), 6.52 (d, J = 8.5 Hz, 1H),6.49-6.43 (m, 1H), 6.40 (dd, J = 8.4, 2.3 Hz, 1H), 5.64 (s, 2H), 5.60(s, 2H), 3.80 (s, 3H), 3.78 (s, 2H), 2.65 (t, J = 5.6 Hz, 2H), 1.56-1.42(m, 2H), 1.29-1.16 (m, 2H), 0.87 (t, J = 7.5 Hz, 3H). 803 4, 6 569 449.11.58 δ 7.74 (s, 1H), 7.33 (br d, J = 8.2 Hz, 2H), 6.97 (d, J = 8.2 Hz,2H), 6.82 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 6.47-6.41 (m,1H), 5.68 (s, 2H), 4.48 (br s, 2H), 3.73 (s, 3H), 3.64-3.53 (m, 2H),1.59 (dt, J = 14.6, 7.3 Hz, 2H), 1.27 (dt, J = 14.9, 7.4 Hz, 2H), 0.90(t, J = 7.3 Hz, 3H) 804 2 43.4 406.4 0.91 δ 8.90 (s, 1H), 8.26 (s, 1H),7.95 (s, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.64 (s, 1H), 7.50 (s, 1H),7.15 (s, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 5.71(s, 2H), 5.33 (s, 2H), 3.77 (s, 3H), 2.90 (s, 1H), 2.73 (d, J = 9.5 Hz,1H), 1.56 (p, J = 7.3 Hz, 2H), 1.23 (q, J = 7.3 Hz, 2H), 0.85 (t, J =7.4 Hz, 3H) 805 2 41.4 427.5 0.94 δ 8.18 (s, 1H), 7.95 (s, 1H), 7.75 (s,1H), 7.26 (s, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H),5.73 (s, 2H), 4.13 (s, 2H), 3.79 (s, 3H), 3.56 (d, J = 7.0 Hz, 1H), 2.90(s, 1H), 2.72 (d, J = 17.1 Hz, 3H), 1.58 (p, J = 7.5 Hz, 2H), 1.28 (dd,J = 14.9, 7.5 Hz, 2H), 1.16 (s, 5H), 0.89 (t, J = 7.4 Hz, 3H) 806 2 41.4453.5 0.82 δ 8.28 (s, 1H), 7.82 (s, 1H), 7.76 (d, J = 12.9 Hz, 1H), 7.21(s, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H), 5.74 (s,2H), 4.13 (s, 1H), 3.78 (s, 3H), 3.58 (q, J = 6.7 Hz, 1H), 3.42 (s, 0H),2.96 (s, 1H), 2.72 (s, 1H), 2.08 (d, J = 12.0 Hz, 2H), 1.89 (d, J = 12.8Hz, 2H), 1.59 (h, J = 7.6 Hz, 2H), 1.45-1.23 (m, 4H), 1.16 (q, J = 11.1Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H) 807 2 2548 439.5 0.97 δ 7.55 (s, 1H),7.03 (s, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.45 (d, J = 7.8 Hz, 1H), 6.39(s, 1H), 5.61 (d, J = 13.7 Hz, 3H), 3.89 (d, J = 19.6 Hz, 3H), 3.84 (s,2H), 3.40 (d, J = 6.7 Hz, 1H), 3.18 (s, 1H), 2.44 (s, 1H), 2.23 (d, J =8.6 Hz, 2H), 1.86 (s, 1H), 1.75 (s, 1H), 1.48 (t, J = 7.4 Hz, 2H), 1.41(d, J = 9.6 Hz, 2H), 1.20 (q, J = 7.5 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H)808 4, 6 157 506.2 1.22 δ 7.74 (br s, 1H), 7.51 (br d, J = 8.5 Hz, 2H),7.05 (br d, J = 8.5 Hz, 2H), 6.84-6.78 (m, 2H), 6.47 (dd, J = 7.8, 2.0Hz, 1H), 5.70 (s, 2H), 4.14 (br s, 2H), 3.75 (s, 3H), 3.61-3.57 (m, 2H),3.12- 3.06 (m, 2H), 1.62-1.56 (m, 2H), 1.32-1.26 (m, 2H), 0.90 (br t, J= 7.3 Hz, 3H). 809 4, 6 74.5 531.2 1.43 δ 7.76 (s, 1H), 7.37-7.31 (m,2H), 7.02-6.94 (m, 2H), 6.85 (d, J = 7.9 Hz, 1H), 6.77 (s, 1H),6.46-6.41 (m, 1H), 5.72-5.69 (m, 2H), 3.73 (s, 3H), 3.62-3.58 (m, 2H),1.64-1.57 (m, 2H), 1.32-1.26 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). 810 4, 6245 561.4 1.0 δ 7.74 (s, 1H), 7.38-7.23 (m, 2H), 6.98 (br d, J = 8.7 Hz,2H), 6.82 (d, J = 8.5 Hz, 1H), 6.75 (s, 1H), 6.42 (br d, J = 7.0 Hz,1H), 5.68 (s, 2H), 3.50 (s, 3H), 3.17 (s, 2H), 3.08 (br dd, J = 6.3, 2.2Hz, 2H), 2.97-2.88 (m, 6H), 2.65 (br s, 2H), 1.63-1.52 (m, 2H),1.28-1.24 (m, 2H), 0.93- 0.81 (m, 3H). 811 2 138 425.2 0.8 δ 7.54 (s,1H), 7.00 (s, 1H), 6.77-6.72 (m, 1H), 6.49-6.40 (m, 2H), 5.59 (d, J =9.5 Hz, 3H), 4.23 (t, J = 6.1 Hz, 1H), 3.82 (s, 3H), 3.38 (q, J = 6.5Hz, 2H), 3.18 (d, J = 13.2 Hz, 2H), 1.95 (t, J = 5.8 Hz, 2H), 1.89 (dd,J = 7.5, 4.9 Hz, 2H), 1.84 (s, 8H), 1.50-1.43 (m, 2H), 1.18 (q, J = 7.4Hz, 2H), 0.84 (t, J = 7.3 Hz, 3H) 812 2 102 450.5 0.77 δ 8.32 (s, 1H),7.95 (s, 1H), 7.77 (s, 1H), 7.15 (s, 1H), 6.96 (d, J = 7.7 Hz, 1H), 6.86(d, J = 7.6 Hz, 1H), 5.74 (s, 2H), 4.22 (s, 1H), 3.78 (s, 2H), 2.90 (s,3H), 2.55 (s, 1H), 1.58 (p, J = 7.3 Hz, 2H), 1.40 (s, 1H), 1.26 (q, J =7.5 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H) 813 2 23.8 424.5 0.78 δ 8.28 (s,1H), 7.96 (s, 1H), 7.90 (s, 0H), 7.76 (s, 1H), 7.27 (s, 0H), 7.17 (s,0H), 7.06 (d, J = 13.9 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.81 (d, J =7.6 Hz, 1H), 5.72 (s, 2H), 3.77 (s, 2H), 3.73 (s, 1H), 3.58 (d, J = 6.9Hz, 1H), 3.17 (d, J = 6.4 Hz, 2H), 2.90 (s, 3H), 2.55 (s, 1H), 1.58 (p,J = 7.1 Hz, 2H), 1.26 (q, J = 7.5 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H) 8142 97.6 411.5 0.83 δ 8.28 (s, 1H), 7.84 (s, 1H), 7.76 (d, J = 9.0 Hz,1H), 7.19 (s, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H),5.74 (s, 2H), 4.66 (t, J = 7.4 Hz, 2H), 4.56 (t, J = 6.6 Hz, 2H), 4.36(q, J = 6.5 Hz, 1H), 4.23 (d, J = 14.0 Hz, 1H), 4.07 (s, 1H), 3.79 (s,3H), 3.58 (d, J = 6.9 Hz, 2H), 2.51 (s, 8H), 1.60 (q, J = 7.4 Hz, 2H),1.28 (dt, J = 15.3, 7.6 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H) 815 2 33.2439.5 1.1 δ 8.29 (d, J = 5.7 Hz, 1H), 7.96 (s, 1H), 7.89 (s, 1H), 7.77(d, J = 5.7 Hz, 1H), 7.15 (s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.85 (d, J= 7.7 Hz, 1H), 5.75 (d, J = 10.6 Hz, 2H), 4.35 (s, 1H), 4.29 (s, 1H),4.25 (s, 1H), 4.07 (s, 1H), 4.00 (s, 1H), 3.79 (d, J = 5.5 Hz, 3H), 3.57(q, J = 6.7 Hz, 1H), 3.43 (s, 1H), 2.90 (s, 2H), 2.73 (d, J = 9.7 Hz,3H), 2.55 (s, 1H), 1.73 (s, 1H), 1.58 (p, J = 7.4 Hz, 2H), 1.27 (q, J =7.4 Hz, 2H), 0.88 (td, J = 7.3, 4.3 Hz, 3H) 816 2 92.5 399.4 1.0 δ 8.19(s, 1H), 7.95 (s, 1H), 7.74 (s, 1H), 7.22 (s, 1H), 7.00 (d, J = 7.8 Hz,1H), 6.86 (d, J = 7.7 Hz, 1H), 5.73 (s, 2H), 4.14 (s, 2H), 3.90 (s, 1H),3.78 (s, 3H), 3.66 (s, 1H), 3.57 (s, 1H), 3.46 (s, 1H), 3.18 (s, 1H),2.95 (t, J = 5.5 Hz, 2H), 2.90 (s, 1H), 2.74 (s, 1H), 2.55 (s, 1H), 1.58(p, J = 7.3 Hz, 2H), 1.28 (h, J = 7.4 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H)817 3 27.8 400.4 1.17 δ 7.73 (s, 1H), 7.64 (s, 1H), 7.33 (d, J = 8.5 Hz,1H), 6.98 (s, 1H), 6.80 (d, J = 7.9 Hz, 1H), 6.64 (d, J = 7.6 Hz, 1H),5.72 (d, J = 16.4 Hz, 1H), 5.64 (d, J = 16.3 Hz, 1H), 4.49 (d, J = 6.4Hz, 1H), 4.44 (s, 2H), 3.87 (s, 1H), 3.75 (s, 3H), 3.46 (d, J = 11.3 Hz,1H), 3.38 (d, J = 6.7 Hz, 1H), 3.14 (s, 1H), 2.52 (s, 2H), 1.68 (dq, J =12.5, 6.6, 6.2 Hz, 2H), 1.47 (t, J = 5.8 Hz, 2H), 1.09 (q, J = 9.0, 8.2Hz, 2H), 0.78 (t, J = 7.3 Hz, 3H) 818 2 258 356.4 1.2 8.13 (s, 1H), 7.73(s, 2H), 6.99 (s, 1H), 6.86- 6.76 (m, 2H), 5.69 (s, 2H), 4.47 (s, 2H),3.76 (s, 3H), 3.57 (d, J = 6.7 Hz, 1H), 1.57 (p, J = 7.2 Hz, 2H), 1.27(dt, J = 15.0, 7.5 Hz, 2H), 1.17 (t, J = 7.2 Hz, 1H), 0.89 (t, J = 7.4Hz, 3H) 819 2 44.4 436.5 0.97 δ 8.27 (t, J = 5.7 Hz, 1H), 7.94 (s, 1H),7.76 (d, J = 0.9 Hz, 1H), 7.30 (s, 1H), 7.19 (s, 0H), 7.09 (s, 1H), 6.93(d, J = 7.8 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 5.71 (s, 2H), 4.30 (s,1H), 4.01 (s, 1H), 3.94 (s, 1H), 3.76 (s, 3H), 3.57 (s, 1H), 3.19 (d, J= 15.2 Hz, 1H), 3.02 (s, 1H), 2.89 (s, 1H), 2.73 (s, 1H), 2.55 (s, 3H),2.22 (s, 1H), 1.84 (d, J = 11.5 Hz, 1H), 1.57 (p, J = 7.2 Hz, 2H), 1.25(h, J = 7.5 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H) 820 2 121 453.5 1.09 δ7.72 (s, 1H), 7.21 (s, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.91 (d, J = 7.7Hz, 1H), 5.71 (s, 2H), 4.07 (s, 2H), 3.78 (s, 3H), 3.54 (s, 1H), 2.89(s, 1H), 2.73 (s, 1H), 2.55 (s, 4H), 1.82-1.71 (m, 5H), 1.59 (dd, J =15.5, 7.8 Hz, 4H), 1.30 (q, J = 7.5 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H)821 2 33.8 438.5 1.27 δ 8.30 (s, 1H), 7.94 (s, 1H), 7.77 (q, J = 1.5 Hz,1H), 7.16 (s, 1H), 6.97 (d, J = 7.7 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H),5.73 (s, 2H), 4.14 (s, 1H), 3.78 (d, J = 1.6 Hz, 3H), 3.57 (s, 1H), 3.23(s, 2H), 2.89 (d, J = 1.5 Hz, 3H), 2.73 (d, J = 1.3 Hz, 3H), 2.55 (s,2H), 2.03 (s, 2H), 1.57 (p, J = 7.3 Hz, 3H), 1.25 (q, J = 7.4 Hz, 3H),0.87 (t, J = 7.3 Hz, 3H) 822 2 244 475.5 1.27 δ 9.95 (s, 0H), 8.20 (d, J= 6.0 Hz, 1H), 7.73 (s, 1H), 7.49 (s, 1H), 7.29 (s, 1H), 7.18 (s, 1H),7.03 (s, 1H), 6.89-6.83 (m, 2H), 6.46 (d, J = 8.3 Hz, 1H), 5.81 (s, 1H),5.66 (s, 1H), 4.20 (s, 1H), 3.86 (s, 1H), 3.72 (s, 1H), 3.46 (t, J = 7.3Hz, 1H), 2.89 (s, 1H), 2.73 (s, 0H), 2.55 (s, 3H), 1.59-1.52 (m, 2H),1.24 (q, J = 7.4 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H) 823 2 21.5 468.6 1.01δ 7.56 (s, 1H), 6.96 (s, 1H), 6.74 (d, J = 7.5 Hz, 1H), 6.43 (dd, J =12.7, 6.8 Hz, 2H), 5.70 (s, 1H), 5.61 (s, 2H), 3.88-3.74 (m, 5H), 3.39(d, J = 10.3 Hz, 2H), 2.59-2.53 (m, 9H), 2.40 (t, J = 6.3 Hz, 4H), 1.91(d, J = 1.2 Hz, 3H), 1.45 (p, J = 7.2 Hz, 2H), 1.16 (h, J = 7.5 Hz, 2H),0.83 (t, J = 7.4 Hz, 3H) 824 2 304 436.5 1.02 δ 7.55 (s, 1H), 6.90 (d, J= 1.5 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.42 (d, J = 7.6 Hz, 1H), 5.61(d, J = 17.6 Hz, 3H), 3.83 (s, 2H), 3.72 (s, 1H), 3.39 (d, J = 6.1 Hz,1H), 3.20 (s, 2H), 2.55 (s, 5H), 1.87 (s, 4H), 1.52-1.42 (m, 2H), 1.19(h, J = 7.3 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H) 825 2 43.5 427.5 1.25 δ8.24 (s, 1H), 7.94 (s, 1H), 7.81 (s, 1H), 7.76 (s, 1H), 7.21 (s, 1H),7.00 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H), 5.74 (s, 2H), 4.25(s, 1H), 3.89 (s, 1H), 3.78 (s, 3H), 3.56 (d, J = 6.9 Hz, 1H), 3.43 (s,1H), 3.17 (s, 1H), 3.08 (s, 1H), 2.89 (s, 1H), 2.73 (s, 1H), 2.66 (s,3H), 2.54 (s, 3H), 1.83 (s, 1H), 1.57 (q, J = 7.3 Hz, 2H), 1.25 (dt, J =14.9, 7.5 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H) 826 2 182 413.5 0.85 δ 8.29(s, 1H), 7.95 (s, 1H), 7.75 (s, 1H), 7.21 (s, 1H), 7.00 (d, J = 7.6 Hz,1H), 6.87 (d, J = 7.7 Hz, 1H), 5.73 (s, 2H), 4.13 (s, 2H), 3.78 (s, 2H),3.58 (t, J = 4.9 Hz, 2H), 3.29 (s, 2H), 3.07 (t, J = 5.1 Hz, 2H), 2.90(s, 2H), 2.74 (s, 2H), 1.59 (p, J = 7.4 Hz, 2H), 1.28 (h, J = 7.8 Hz,2H), 0.89 (t, J = 7.4 Hz, 3H) 827 2 47.6 438.5 0.89 δ 7.65 (s, 1H), 6.94(d, J = 7.9 Hz, 2H), 6.82 (d, J = 7.6 Hz, 1H), 5.74 (s, 1H), 5.35 (s,2H), 3.87 (d, J = 2.1 Hz, 2H), 3.84 (s, 9H), 3.40 (s, 2H), 2.54 (s, 3H),2.14 (s, 3H), 1.83 (s, 1H), 1.52 (t, J = 7.6 Hz, 2H), 1.28 (q, J = 7.5Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H) 828 2 98.7 411.5 0.92 δ 8.17 (s, 0H),7.72 (d, J = 2.4 Hz, 1H), 7.63 (s, 0H), 7.24 (d, J = 4.4 Hz, 1H), 7.03(d, J = 7.7 Hz, 1H), 6.92 (d, J = 7.7 Hz, 1H), 5.73 (s, 2H), 4.09 (s,2H), 3.78 s, 3H), 3.61-3.54 (m, 1H), 3.48 (s, 2H), 2.93 (s, 1H), 2.55(s, 1H), 1.62 (q, J = 7.4 Hz, 2H), 1.38-1.27 (m, 11H), 1.21- 1.14 (m,1H), 0.92 (t, J = 7.4 Hz, 3H) 829 15 1336 448.2 δ 8.15 (s, 1H), 7.82 (s,1H), 7.74 (br d, J = 7.9 Hz, 1H), 7.59 (br s, 1H), 7.47 (d, J = 8.5 Hz,2H), 7.05-6.93 (m, 3H), 5.53 (br s, 1H), 5.42 (s, 2H), 1.55 (br t, J =7.5 Hz, 2H), 1.43 (s, 6H), 1.36-1.25 (m, 2H), 0.89 (br t, J = 7.5 Hz,3H) 830 2 30.2 423.5 0.92 δ 8.25 (s, 1H), 7.83 (s, 1H), 7.77 (s, 1H),7.21 (s, 1H), 6.99 (d, J = 7.7 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 5.76(s, 2H), 4.23 (s, 1H), 3.80 (s, 3H), 3.57 (d, J = 6.6 Hz, 1H), 2.93 (s,1H), 2.55 (s, 4H), 1.78 (s, 1H), 1.65 (s, 1H), 1.58 (q, J = 7.3 Hz, 3H),1.25 (q, J = 7.4 Hz, 2H), 1.17 (t, J = 7.2 Hz, 1H), 0.88 (t, J = 7.3 Hz,3H) 831 2 104 411.5 1.0 δ 7.74 (s, 1H), 7.21 (s, 1H), 6.99 (d, J = 8.0Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 5.74 (s, 1H), 4.12 (s, 1H), 3.78 (s,3H), 3.57 (d, J = 6.6 Hz, 1H), 3.41 (s, 1H), 2.93 (s, 3H), 2.89 (s, 1H),2.55 (s, 3H), 1.60 (q, J = 7.5 Hz, 3H), 1.30 (dq, J = 15.5, 7.5 Hz, 3H),1.17 (t, J = 7.3 Hz, 6H), 0.89 (q, J = 7.2 Hz, 4H) 832 2 141 425.5 0.82δ 8.31 (t, J = 5.6 Hz, 1H), 7.77 (d, J = 0.8 Hz, 1H), 7.19 (s, 1H), 6.99(d, J = 7.7 Hz, 1H), 6.85 (d, J = 7.7 Hz, 1H), 5.75 (s, 2H), 4.24 (s,1H), 3.79 (s, 3H), 3.58 (t, J = 6.7 Hz, 1H), 3.09 (s, 2H), 2.97-2.88 (m,1H), 2.55 (d, J = 0.8 Hz, 9H), 1.57 (p, J = 7.3 Hz, 2H), 1.25 (h, J =7.3 Hz, 2H), 1.17 (t, J = 7.3 Hz, 1H), 0.88 (t, J = 7.3 Hz, 3H) 833 231.1 409.5 1.11 δ 8.22 (s, 1H), 7.76 (s, 1H), 7.22 (s, 1H), 7.01 (d, J =7.5 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H), 5.75 (s, 2H), 4.31 (s, 2H), 3.80(s, 3H), 3.61- 3.54 (m, 1H), 2.93 (s, 1H), 2.55 (s, 8H), 1.57 (p, J =7.2 Hz, 2H), 1.27 (dt, J = 14.7, 7.5 Hz, 2H), 1.17 (t, J = 7.2 Hz, 1H),0.88 (t, J = 7.3 Hz, 3H) 834 3 29.9 494.3 0.89 δ 7.79 (s, 1H), 7.60 (d,J = 8.5 Hz, 1H), 7.13 (s, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.70 (d, J =7.7 Hz, 1H), 5.82-5.69 (m, 2H), 4.52 (d, J = 9.2 Hz, 1H), 3.80 (s, 2H),3.52 (s, 2H), 3.38 (t, J = 6.5 Hz, 2H), 2.92 (d, J = 6.0 Hz, 1H), 2.55(s, 10H), 1.71 (q, J = 6.3 Hz, 2H), 1.53 (d, J = 8.7 Hz, 2H), 1.16 (td,J = 8.1, 7.7, 2.7 Hz, 4H), 0.81 (t, J = 7.3 Hz, 3H) 835 3 89 480.9 1 δ7.57 (s, 1H), 6.91 (s, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.35 (d, J = 7.7Hz, 1H), 5.71-5.61 (m, 3H), 5.55 (d, J = 17.0 Hz, 1H), 4.31 (d, J = 9.9Hz, 1H), 3.84 (s, 2H), 3.79 (s, 2H), 3.44 (s, 1H), 3.29 (d, J = 7.1 Hz,1H), 3.26 (d, J = 48.2 Hz, 5H), 3.17 (s, 0H), 2.55 (s, 6H), 1.84 (s,5H), 1.63 (dd, J = 13.7, 6.6 Hz, 1H), 1.53-1.47 (m, 1H), 1.37 (dt, J =34.6, 7.6 Hz, 2H), 1.08-1.01 (m, 2H), 0.76 (t, J = 7.3 Hz, 3H) 836 315.3 451.2 0.83 δ 8.63 (s, 1H), 7.76 (s, 1H), 7.52 (d, J = 7.2 Hz, 2H),7.35 (s, 1H), 7.14 (s, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.69 (d, J = 7.8Hz, 1H), 5.77 (d, J = 16.5 Hz, 1H), 5.69 (d, J = 16.4 Hz, 1H), 5.29 (s,2H), 4.52 (d, J = 7.0 Hz, 1H), 3.79 (s, 3H), 3.38 (d, J = 6.7 Hz, 1H),3.18 (s, 0H), 2.93 (s, 2H), 2.55 (s, 6H), 1.71 (p, J = 6.1 Hz, 2H), 1.50(q, J = 7.2 Hz, 2H), 1.13 (dt, J = 35.1, 7.5 Hz, 5H), 0.78 (t, J = 7.3Hz, 3H) 837 3 2.4 491.2 0.71 δ 8.67 (d, J = 5.1 Hz, 2H), 7.91 (s, 0H),7.78 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.53 (d, J = 5.1 Hz, 2H), 7.22(s, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 5.82-5.71(m, 2H), 4.56 (d, J = 7.2 Hz, 1H), 4.22 (d, J = 19.9 Hz, 4H), 3.79 (s,3H), 3.41 (d, J = 6.3 Hz, 0H), 2.93 (q, J = 6.7 Hz, 1H), 2.55 (s, 0H),1.79-1.72 (m, 2H), 1.56 (p, J = 8.0, 7.4 Hz, 2H), 1.23 (d, J = 10.9 Hz,1H), 1.18 (q, J = 8.3, 7.4 Hz, 4H), 0.84 (t, J = 7.3 Hz, 3H) 838 3 39.6483.1 0.7 δ 7.80 (s, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.19 (s, 1H), 6.95(d, J = 7.9 Hz, 1H), 6.68 (d, J = 7.7 Hz, 1H), 5.79 (d, J = 16.8 Hz,1H), 5.74 (d, J = 16.7 Hz, 1H), 4.57-4.51 (m, 1H), 3.81 (s, 3H), 3.40(d, J = 17.5 Hz, 1H), 3.22 (s, 1H), 2.97- 2.89 (m, 2H), 2.55 (s, 1H),2.02 (s, 1H), 1.79- 1.68 (m, 2H), 1.56-1.50 (m, 2H), 1.16 (dt, J = 12.8,7.3 Hz, 5H), 0.82 (t, J = 7.3 Hz, 3H) 839 3 242 481.1 0.74 δ 7.57 (s,1H), 7.01 (s, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.37 (d, J = 7.7 Hz, 1H),5.74-5.66 (m, 1H), 5.64 (s, 2H), 5.55 (d, J = 17.1 Hz, 1H), 4.31 (s,1H), 3.84 (s, 2H), 3.63 (d, J = 8.6 Hz, 7H), 3.60 (d, J = 6.0 Hz, 6H),3.37 (s, 1H), 3.30 (q, J = 7.9, 7.1 Hz, 2H), 3.16 (d, J = 12.7 Hz, 1H),2.81 (s, 1H), 2.70 (d, J = 10.3 Hz, 1H), 2.55 (s, 7H), 1.81 (s, 3H),1.63 (dd, J = 13.1, 6.4 Hz, 1H), 1.51 (dd, J = 13.3, 6.3 Hz, 2H), 1.41(d, J = 6.8 Hz, 1H), 1.34 (t, J = 7.4 Hz, 1H), 1.03 (d, J = 8.0 Hz, 2H),0.75 (t, J = 7.3 Hz, 3H) 840 3 14.7 484.2 0.82 δ 8.97 (s, 2H), 7.85 (s,2H), 7.77 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 1.5 Hz, 1H),7.02 (dd, J = 7.7, 1.5 Hz, 1H), 5.76 (d, J = 2.9 Hz, 2H), 4.57 (dq, J =14.2, 6.8 Hz, 2H), 4.17 (s, 2H), 3.93 (dd, J = 11.3, 3.9 Hz, 2H), 3.80(s, 3H), 3.53-3.16 (m, 5H), 2.07-1.92 (m, 2H), 1.75 (q, J = 6.6 Hz, 2H),1.68-1.46 (m, 4H), 1.21 (h, J = 7.3 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H)841 3 9.7 454.2 0.83 δ 7.92 (s, 1H), 7.56 (s, 1H), 7.05 (s, 1H), 6.78(d, J = 7.9 Hz, 1H), 6.40 (d, J = 7.8 Hz, 1H), 5.73 (d, J = 8.9 Hz, 0H),5.64 (d, J = 18.8 Hz, 2H), 5.55 (d, J = 17.0 Hz, 1H), 4.30 (s, 1H), 3.84(s, 1H), 3.76-3.56 (m, 3H), 3.29 (t, J = 6.5 Hz, 2H), 3.21-3.15 (m, 1H),2.89 (s, 1H), 2.73 (s, 1H), 2.55 (s, 1H), 2.03 (d, J = 9.1 Hz, 2H), 1.74(t, J = 9.7 Hz, 2H), 1.62 (t, J = 10.0 Hz, 2H), 1.52 (dt, J = 16.0, 8.1Hz, 2H), 1.37 (dt, J = 33.8, 7.5 Hz, 2H), 1.09-1.02 (m, 2H), 0.75 (t, J= 7.3 Hz, 3H) 842 3 28.3 484.3 0.77 δ 7.74 (s, 1H), 7.41 (s, 1H), 7.08(s, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.71 (d, J = 7.9 Hz, 1H), 5.74 (d, J= 16.7 Hz, 1H), 5.65 (d, J = 16.5 Hz, 1H), 4.49 (s, 1H), 4.07 (s, 5H),4.05 (s, 9H), 3.78 (s, 3H), 3.38 (t, J = 6.1 Hz, 4H), 2.89 (s, 1H), 2.83(s, 1H), 2.55 (s, 13H), 1.71 (s, 2H), 1.65 (d, J = 6.2 Hz, 1H),1.50-1.44 (m, 2H), 1.18-1.00 (m, 4H), 0.76 (t, J = 7.3 Hz, 3H) 843 355.9 481.2 1.1 δ 7.57 (s, 1H), 7.01 (s, 1H), 6.78 (d, J = 7.9 Hz, 1H),6.37 (d, J = 7.7 Hz, 1H), 5.74-5.66 (m, 1H), 5.64 (s, 2H), 5.55 (d, J =17.1 Hz, 1H), 4.31 (s, 1H), 3.84 (s, 2H), 3.63 (d, J = 8.6 Hz, 7H), 3.60(d, J = 6.0 Hz, 6H), 3.37 (s, 1H), 3.30 (q, J = 7.9, 7.1 Hz, 2H), 3.16(d, J = 12.7 Hz, 1H), 2.81 (s, 1H), 2.70 (d, J = 10.3 Hz, 1H), 2.55 (s,7H), 1.81 (s, 3H), 1.63 (dd, J = 13.1, 6.4 Hz, 1H), 1.51 (dd, J = 13.3,6.3 Hz, 2H), 1.41 (d, J = 6.8 Hz, 1H), 1.34 (t, J = 7.4 Hz, 1H), 1.03(d, J = 8.0 Hz, 2H), 0.75 (t, J = 7.3 Hz, 3H) 844 3 4.6 517.4 0.73 δ8.27 (s, 1H), 8.20 (d, J = 5.0 Hz, 1H), 7.91 (s, 0H), 7.58 (s, 1H),7.07-7.01 (m, 2H), 6.82 (d, J = 7.8 Hz, 1H), 6.41 (d, J = 7.7 Hz, 1H),5.78 (d, J = 8.5 Hz, 1H), 5.71-5.64 (m, 2H), 5.55 (d, J = 17.1 Hz, 1H),4.30 (s, 1H), 3.80 (d, J = 30.8 Hz, 4H), 3.60 (s, 1H), 3.53 (s, 1H),3.35- 3.29 (m, 2H), 2.88 (s, 1H), 2.77 (d, J = 5.9 Hz, 2H), 2.72 (s,1H), 2.66 (d, J = 6.5 Hz, 2H), 2.55 (s, 1H), 1.90 (s, 4H), 1.63 (d, J =8.6 Hz, 1H), 1.51 (s, 1H), 1.39 (s, 1H), 1.35-1.28 (m, 1H), 1.01 (d, J =12.4 Hz, 2H), 0.71 (t, J = 7.4 Hz, 3H) 845 2 21.2 439.0 0.92 δ 8.29 (s,1H), 7.76 (s, 1H), 7.17 (s, 1H), 6.98 (d, J = 7.8 Hz, 1H), 6.84 (d, J =7.6 Hz, 1H), 5.73 (s, 2H), 4.19 (s, 1H), 3.78 (s, 2H), 3.55 (s, 1H),3.40 (d, J = 33.6 Hz, 1H), 3.23 (s, 1H), 3.16 (s, 1H), 2.99 (s, 1H),2.55 (s, 9H), 2.05 (s, 1H), 1.60-1.53 (m, 2H), 1.25 (q, J = 7.4 Hz, 2H),0.87 (t, J = 7.4 Hz, 3H) 846 5 198 525.2 0.96 δ 7.82 (s, 1H), 7.76 (s,1H), 7.58 (d, J = 8.6 Hz, 1H), 7.20 (s, 1H), 6.99 (d, J = 8.3 Hz, 1H),6.79 (d, J = 7.7 Hz, 1H), 5.82 (d, J = 16.4 Hz, 1H), 5.75 (d, J = 16.6Hz, 1H), 4.49 (s, 1H), 4.13 (s, 2H), 3.95-3.88 (m, 2H), 3.80-3.76 (m,2H), 3.56 (d, J = 29.7 Hz, 1H), 3.29 (t, J = 11.8 Hz, 3H), 2.98 (d, J =6.8 Hz, 2H), 2.92 (s, 1H), 2.51 (s, 8H), 1.99 (d, J = 11.9 Hz, 2H), 1.78(s, 3H), 1.72 (d, J = 8.7 Hz, 1H), 1.62-1.49 (m, 4H), 1.16 (t, J = 7.1Hz, 3H), 0.82 (t, J = 7.2 Hz, 3H) 847 5 187 539.1 0.81 δ 7.89 (d, J =6.5 Hz, 1H), 7.76 (d, J = 1.2 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.18(d, J = 11.6 Hz, 1H), 7.06-6.98 (m, 1H), 6.83 (d, J = 7.8 Hz, 1H), 5.81(d, J = 16.1 Hz, 1H), 5.74 (d, J = 18.4 Hz, 1H), 4.50 (s, 1H), 4.09 (s,2H), 3.83 (d, J = 8.8 Hz, 2H), 3.79-3.72 (m, 9H), 3.69 (s, 1H), 3.47 (t,J = 11.9 Hz, 2H), 2.98 (d, J = 5.8 Hz, 2H), 1.85 (s, 2H), 1.78 (s, 3H),1.73 (d, J = 12.3 Hz, 4H), 1.53 (dt, J = 13.9, 7.5 Hz, 2H), 1.48 (s,3H), 1.18 (q, J = 7.6 Hz, 2H), 0.82 (t, J = 7.3 Hz, 3H) 848 3 33.0 497.91.02 δ 7.57 (s, 1H), 7.12 (s, 1H), 6.86 (d, J = 7.8 Hz, 1H), 6.43 (d, J= 7.8 Hz, 1H), 5.74 (d, J = 8.2 Hz, 1H), 5.69-5.62 (m, 3H), 5.57 (d, J =16.9 Hz, 1H), 4.32 (s, 1H), 3.86 (s, 3H), 3.74-3.66 (m, 3H), 3.55 (s,4H), 3.29 (t, J = 6.5 Hz, 2H), 2.55 (s, 5H), 1.91 (s, 2H), 1.65 (dd, J =13.3, 6.0 Hz, 1H), 1.57 (s, 2H), 1.49 (s, 2H), 1.39 (dq, J = 22.4, 7.8Hz, 3H), 1.17 (s, 3H), 1.07 (q, J = 7.6 Hz, 2H), 0.78 (t, J = 7.3 Hz,3H) 849 13 2030 440.1 0.97 δ 8.45 (d, J = 1.5 Hz, 1H), 7.73 (dd, J =8.1, 2.0 Hz, 1H), 7.61 (br t, J = 4.9 Hz, 1H), 7.55 (s, 1H), 7.18 (d, J= 7.9 Hz, 1H), 5.70 (s, 2H), 5.65 (s, 2H), 3.91 (s, 1H), 3.51-3.31 (m,6H), 2.48- 2.08 (m, 8H), 1.65-1.51 (m, 2H), 1.37-1.26 (m, 2H), 0.91 (t,J = 7.3 Hz, 3H) 850 13 1616 396.2 1.06 δ 8.45 (d, J = 1.5 Hz, 1H), 7.74(dd, J = 7.8, 2.0 Hz, 1H), 7.64-7.57 (m, 1H), 7.55 (s, 1H), 7.18 (d, J =7.9 Hz, 1H), 5.70 (s, 2H), 5.64 (s, 2H), 2.71 (br t, J = 4.4 Hz, 4H),2.30 (br s, 4H), 1.66- 1.51 (m, 2H), 1.37-1.26 (m, 2H), 0.91 (t, J = 7.3Hz, 3H) 851 13 1770 381.1 1.38 δ 8.52 (s, 1H), 7.96 (br d, J = 4.0 Hz,1H), 7.83 (dd, J = 8.0, 1.7 Hz, 1H), 7.61 (s, 1H), 7.25 (d, J = 8.0 Hz,1H), 6.19 (br d, J = 2.1 Hz, 2H), 5.76 (s, 2H), 3.86 (br s, 2H),3.56-3.39 (m, 2H), 2.68 (br s, 4H), 1.76 (br s, 4H), 1.59 (quin, J = 7.3Hz, 2H), 1.36-1.25 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H) 852 13 3170 408.01.24 δ 8.52 (s, 1H), 7.90 (dd, J = 8.1, 1.7 Hz, 1H), 7.78 (s, 1H), 7.48(d, J = 7.9 Hz, 1H), 5.86 (s, 2H), 4.09-3.88 (m, 2H), 3.65-3.55 (m, 2H),3.23-3.08 (m, 4H), 3.07-2.91 (m, 2H), 2.18 (br d, J = 10.7 Hz, 1H), 1.82(br d, J = 11.3 Hz, 1H), 1.64 (quin, J = 7.2 Hz, 2H), 1.32 (sxt, J = 7.4Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H). 853 9 420 397.9 1.35 δ 8.46 (br s,1H), 7.88 (br s, 1H), 7.81 (s, 1H), 7.40 (d, J = 11.2 Hz, 1H), 7.27 (brd, J = 7.6 Hz, 1H), 7.06 (t, J = 7.9 Hz, 1H), 5.90 (s, 2H), 4.04 (s,2H), 3.67 (br t, J = 8.1 Hz, 1H), 3.58 (br d, J = 6.1 Hz, 2H), 2.19-2.10(m, 4H), 2.08 (s, 1H), 1.85-1.69 (m, 2H), 1.60 (quin, J = 7.3 Hz, 2H),1.35-1.21 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H) 854 2 46.8 437.0 0.71 δ 7.55(s, 1H), 6.99 (s, 1H), 6.80-6.74 (m, 1H), 6.43 (d, J = 7.7 Hz, 1H), 5.65(s, 1H), 5.59 (s, 2H), 3.82 (s, 3H), 3.63 (s, 1H), 3.39 (d, J = 6.3 Hz,2H), 3.19 (d, J = 13.3 Hz, 1H), 2.91-2.83 (m, 1H), 2.75-2.67 (m, 1H),2.55 (s, 5H), 1.81 (d, J = 1.7 Hz, 5H), 1.57 (d, J = 10.1 Hz, 1H), 1.46(p, J = 7.2 Hz, 2H), 1.17 (h, J = 7.5 Hz, 2H), 0.83 (t, J = 7.3 Hz, 3H).855 11 640 411.1 1.36 δ 8.03 (t, J = 5.4 Hz, 1H), 8.02 (s, 1H), 7.51 (s,1H), 7.46 (s, 1H), 5.65 (s, 2H), 5.60 (s, 1H), 3.88 (s, 3H), 3.64 (s,1H), 3.18 (s, 2H), 3.16- 3.10 (m, 1H), 2.10-1.99 (m, 2H), 1.92 (s, 3H),1.76-1.45 (m, 6H), 1.39 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H)856 11 1160 441.3 1.28 δ 8.06 (d, J = 1.6 Hz, 1H), 7.56 (d, J = 1.5 Hz,1H), 7.47 (s, 1H), 5.65 (d, J = 6.9 Hz, 3H), 3.88 (s, 2H), 3.82 (d, J =12.6 Hz, 3H), 3.30-3.20 (m, 2H), 3.18 (s, 1H), 3.00 (s, 1H), 2.64 (s,1H), 2.55 (s, 3H), 1.92 (s, 1H), 1.78 (d, J = 12.8 Hz, 2H), 1.64 (p, J =7.2 Hz, 2H), 1.39 (h, J = 7.4 Hz, 2H), 1.34-1.22 (m, 2H), 0.94 (t, J =7.4 Hz, 3H). 857 4, 6 11.5 519.9 1.48 δ 8.22 (br t, J = 6.0 Hz, 1H),7.76 (s, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 6.86(d, J = 8.2 Hz, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.48 (dd, J = 8.2, 2.1Hz, 1H), 5.71 (s, 2H), 4.14 (s, 2H), 3.74 (s, 3H), 3.59 (q, J = 6.5 Hz,2H), 2.76 (s, 2H), 1.59 (quin, J = 7.3 Hz, 2H), 1.34-1.23 (m, 2H), 1.17(s, 6H), 0.90 (t, J = 7.5 Hz, 3H) 858 4, 6 10.8 532.2 1.38 δ 7.55 (s,1H), 7.34 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.75 (d, J =1.8 Hz, 1H), 6.52 (d, J = 8.2 Hz, 1H), 6.46 (br t, J = 5.2 Hz, 1H), 6.39(dd, J = 8.2, 2.1 Hz, 1H), 5.64 (s, 2H), 5.60 (s, 2H), 3.71 (s, 3H),3.42 (br d, J = 5.8 Hz, 2H), 3.26 (td, J = 11.4, 2.0 Hz, 4H), 2.63-2.57(m, 1H), 1.80-1.73 (m, 2H), 1.55-1.46 (m, 2H), 1.32-1.18 (m, 4H), 0.87(t, J = 7.3 Hz, 3H). 859 4, 6 13.1 531.9 1.41 δ 7.55 (s, 1H), 7.26 (d, J= 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 2.1 Hz, 1H), 6.53(d, J = 8.2 Hz, 1H), 6.47 (br t, J = 4.0 Hz, 1H), 6.40 (dd, J = 8.5, 2.1Hz, 1H), 5.64 (s, 2H), 5.60 (s, 2H), 3.80 (s, 3H), 3.46-3.35 (m, 1H),2.69- 2.60 (m, 2H), 2.02 (br t, J = 10.4 Hz, 2H), 1.72- 1.64 (m, 2H),1.50 (quin, J = 7.2 Hz, 2H), 1.42- 1.33 (m, 2H), 1.22 (dq, J = 14.8, 7.4Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H). 860 4, 6 19.5 502.2 1.2 δ 7.55 (s,1H), 7.32 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.75 (d, J =1.9 Hz, 1H), 6.55- 6.43 (m, 2H), 6.39 (dd, J = 8.5, 2.0 Hz, 1H), 5.65(s, 2H), 5.60 (s, 2H), 3.79 (s, 3H), 3.61 (s, 2H), 3.18 (br d, J = 4.7Hz, 1H), 2.06 (br d, J = 7.7 Hz, 2H), 1.78-1.46 (m, 6H), 1.27-1.18 (m,2H), 0.87 (t, J = 7.3 Hz, 3H). 861 3 1.2 470.3 0.82 δ 7.90-7.82 (m, 2H),7.78 (s, 1H), 7.62-7.55 (m, 1H), 7.17 (s, 1H), 6.95 (br d, J = 7.0 Hz,1H), 6.68 (d, J = 7.6 Hz, 1H), 5.84-5.70 (m, 2H), 4.64-4.55 (m, 1H),4.54-4.48 (m, 1H), 4.32 (s, 2H), 4.25-4.21 (m, 2H), 4.20 (br s, 1H),3.91 (br d, J = 6.3 Hz, 2H), 3.80 (s, 3H), 3.23 (s, 3H), 1.75-1.68 (m,2H), 1.57-1.48 (m, 2H), 1.15 (sxt, J = 7.4 Hz, 2H), 0.81 (t, J = 7.3 Hz,3H). 862 3 11.0 476.04 0.86 δ 7.57-7.55 (m, 1H), 6.98 (s, 1H), 6.76 (d,J = 7.7 Hz, 1H), 6.38 (d, J = 8.0 Hz, 1H), 5.71 (br d, J = 8.5 Hz, 1H),5.68-5.62 (m, 3H), 5.59- 5.52 (m, 1H), 4.45-4.37 (m, 1H), 4.36-4.27 (m,1H), 3.85 (s, 3H), 3.66 (s, 2H), 3.56 (br t, J = 12.5 Hz, 4H), 3.33-3.28(m, J = 6.3 Hz, 2H), 1.68-1.58 (m, 1H), 1.54-1.46 (m, 1H), 1.45- 1.30(m, 2H), 1.09-1.00 (m, 2H), 0.75 (t, J = 7.4 Hz, 3H) 863 3 9.5 456.20.77 δ 7.58-7.56 (m, 1H), 6.94 (s, 1H), 6.71 (br d, J = 7.6 Hz, 1H),6.36 (d, J = 7.7 Hz, 1H), 5.70- 5.65 (m, 2H), 5.65 (s, 2H), 5.58-5.52(m, 1H), 5.35-5.29 (m, 1H), 4.32 (br dd, J = 8.3, 4.3 Hz, 1H), 4.16 (brd, J = 5.1 Hz, 1H), 3.85 (s, 3H), 3.50 (s, 2H), 3.48-3.46 (m, 2H),3.33-3.31 (m, 2H), 2.72 (br t, J = 6.0 Hz, 2H), 1.68-1.59 (m, 1H),1.55-1.46 (m, 1H), 1.45-1.38 (m, 1H), 1.37-1.30 (m, 1H), 1.09-0.99 (m,2H), 0.76 (t, J = 7.2 Hz, 3H) 864 7 216 426.17 0.8 δ 7.56-7.52 (m, 1H),7.01 (s, 1H), 6.77 (br d, J = 7.6 Hz, 1H), 6.57 (br d, J = 7.6 Hz, 1H),5.66 (s, 2H), 5.55 (s, 2H), 3.83 (s, 3H), 3.63-3.54 (m, 2H), 3.19-3.11(m, 1H), 2.07-2.00 (m, 2H), 1.75-1.65 (m, 2H), 1.65-1.56 (m, 1H),1.56-1.46 (m, 1H), 1.05 (t, J = 6.9 Hz, 3H) 865 7 959 412.2 0.90 δ7.56-7.52 (m, 1H), 6.99 (s, 1H), 6.76 (br d, J = 7.6 Hz, 1H), 6.62-6.53(m, 1H), 5.65 (s, 2H), 5.54 (s, 2H), 3.82 (s, 3H), 3.58 (q, J = 5.8 Hz,1H), 3.21 (s, 2H), 3.13-3.05 (m, 1H), 2.06- 1.98 (m, 2H), 1.70-1.61 (m,2H), 1.60-1.55 (m, 1H), 1.54-1.45 (m, 1H) 866 8 19.5 510.23 1.23 δ9.28-9.02 (m, 1H), 7.84-7.77 (m, 1H), 7.75 (s, 1H), 7.68-7.59 (m, 1H),7.19 (s, 1H), 6.96 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 5.80-5.66 (m, 2H), 4.60-4.49 (m, 1H), 3.99 (br s, 2H), 3.78 (s, 3H),3.42-3.40 (m, 2H), 2.99- 2.97 (m, 1H), 2.22 (br d, J = 1.7 Hz, 1H),2.07- 2.00 (m, 1H), 1.84-1.80 (m, 1H), 1.76-1.71 (m, 1H), 1.57-1.51 (m,1H), 1.21-1.13 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H), 0.80-0.75 (m, 9H) 86712 302 470.4 0.71 δ 8.96 (s, 1H), 7.85 (s, 1H), 7.78 (s, 1H), 7.73 (d, J= 8.1 Hz, 1H), 7.19 (d, J = 19.6 Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 6.84(d, J = 7.7 Hz, 1H), 5.82-5.70 (m, 2H), 4.46 (m, 1H), 4.16 (s, 2H), 3.94(d, J = 11.4 Hz, 2H), 3.80 (s, 2H), 3.56 (s, 1H), 3.31 (t, J = 11.7 Hz,3H), 2.00 (d, J = 12.5 Hz, 2H), 1.76 (p, J = 6.9 Hz, 2H), 1.62 (tt, J =13.2, 5.8 Hz, 4H), 0.80 (t, J = 7.4 Hz, 3H) 868 3 1.4 498.21 0.95 δ7.59-7.51 (m, 1H), 6.94 (s, 1H), 6.79-6.66 (m, 1H), 6.36 (d, J = 7.7 Hz,1H), 5.69-5.65 (m, 1H), 5.63 (s, 2H), 5.55-5.50 (m, 1H), 4.34- 4.27 (m,1H), 4.06 (quin, J = 6.1 Hz, 1H), 3.84 (s, 3H), 3.51-3.49 (m, 4H), 3.30(m, 2H), 2.78- 2.73 (m, 2H), 1.90 (s, 1H), 1.66-1.59 (m, 1H), 1.54-1.45(m, 1H), 1.43-1.36 (m, 1H), 1.36- 1.29 (m, 1H), 1.05-1.03 (m, 2H), 1.02(s, 3H), 1.01 (s, 3H), 0.74 (t, J = 7.3 Hz, 3H) 869 12 897 484.1 1.13 δ7.59 (s, 1H), 7.08 (s, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.30 (d, J = 7.8Hz, 1H), 5.67 (d, J = 17.3 Hz, 1H), 5.62 (d, J = 10.8 Hz, 2H), 5.55 (d,J = 17.2 Hz, 1H), 5.43 (d, J = 8.9 Hz, 1H), 4.16 (s, 1H), 3.89 (m, 4H),3.82 (s, 3H), 3.76 (s, 1H), 3.70 (s, 1H), 3.25-3.15 (m, 4H), 1.88 (s,1H), 1.73 (m, 3H), 1.62 (m, 2H), 1.52 (m, 1H), 1.23 (m, 2H), 0.67 (dd, J= 6.9, 2.8 Hz, 6H) 870 9 1796 457.3 1.23 δ 8.45 (br t, J = 5.6 Hz, 1H),7.81 (s, 1H), 7.21 (d, J = 11.0 Hz, 1H), 7.16-7.07 (m, 1H), 6.96 (t, J =7.9 Hz, 1H), 5.87 (s, 2H), 3.72 (t, J = 5.2 Hz, 2H), 3.20-3.10 (m, 2H),2.55 (s, 4H), 1.63- 1.50 (m, 2H), 1.29-1.18 (m, 2H), 0.87 (t, J = 7.3Hz, 3H) 871 12 61 469.9 1.04 δ 7.57 (s, 1H), 7.08 (s, 1H), 6.81 (d, J =7.7 Hz, 1H), 6.48 (d, J = 7.7 Hz, 1H), 5.71 (d, J = 16.9 Hz, 1H), 5.63(s, 2H), 5.48 (d, J = 16.9 Hz, 1H), 4.30-4.18 (m, 1H), 3.85 (s, 3H),3.80 (d, J = 12.0 Hz, 2H), 3.71 (s, 2H), 3.60-3.52 (m, 1H), 3.45-3.33(m, 1H), 3.22 (t, J = 11.7 Hz, 1H), 1.83-1.70 (m, 3H), 1.49 (m, 1H),1.34-1.19 (m, 4H), 1.09-1.03 (m, 2H), 1.03-0.94 (m, 2H), 0.76 (t, J =7.3 Hz, 3H) 872 3 32.7 454.16 1.09 δ 7.57-7.54 (m, 1H), 7.05-7.01 (m,1H), 6.76 (d, J = 7.7 Hz, 1H), 6.40-6.37 (m, 1H), 5.66- 5.64 (m, 1H),5.63-5.61 (m, 2H), 5.57-5.52 (m, 1H), 4.35-4.27 (m, 1H), 3.84 (s, 3H),3.56 (s, 2H), 3.29 (br t, J = 6.5 Hz, 2H), 3.18-3.06 (m, 2H), 2.07-2.00(m, 2H), 1.90 (s, 2H), 1.71- 1.64 (m, 2H), 1.63-1.57 (m, 2H), 1.54-1.46(m, 2H), 1.44-1.37 (m, 1H), 1.36-1.30 (m, 1H), 1.10-1.01 (m, 2H), 0.76(t, J = 7.3 Hz, 3H) 873 10 253 366.3 1.37 δ 7.57 (s, 1H), 7.03 (s, 1H),6.82 (d, J = 7.9 Hz, 1H), 6.59 (br s, 1H), 6.48 (d, J = 7.7 Hz, 1H),5.78 (br s, 2H), 5.63 (s, 2H), 3.98 (s, 2H), 3.84 (s, 3H), 3.38 (br. m,2H), 1.48 (quin, J = 7.3 Hz, 2H), 1.25-1.12 (m, 2H), 0.85 (t, J = 7.3Hz, 3H) 874 14 2048 371.15 1.07 δ 7.32-7.17 (m, 2H), 6.96 (br d, J = 7.2Hz, 2H), 6.48-6.39 (m, 1H), 6.39-6.09 (m, 1H), 5.85- 5.69 (m, 2H),5.39-5.06 (m, 1H), 4.63-4.50 (m, 1H), 4.46-4.40 (m, 2H), 4.39-4.31 (m,1H), 3.36-3.32 (m, 2H), 1.73-1.58 (m, 2H), 1.41 (br d, J = 7.1 Hz, 2H),1.08-0.89 (m, 2H), 0.83-0.66 (m, 3H) 875 14 58 423.91 1.03 δ 7.82-7.78(m, 1H), 7.42 (br d, J = 8.5 Hz, 1H), 7.37 (br d, J = 7.9 Hz, 2H), 7.01(br d, J = 8.2 Hz, 2H), 5.85 (s, 2H), 4.47-4.40 (m, 1H), 3.63- 3.54 (m,1H), 3.28 (br t, J = 6.3 Hz, 2H), 2.16- 2.09 (m, 2H), 2.08-2.00 (m, 2H),1.74 (br dd, J = 15.3, 8.9 Hz, 2H), 1.70-1.62 (m, 2H), 1.50- 1.40 (m,2H), 1.06-0.94 (m, 2H), 0.74 (t, J = 7.3 Hz, 3H) 876 14 1270 453.99 0.98δ 7.63-7.56 (m, 1H), 7.26 (br d, J = 7.9 Hz, 2H), 6.94 (br d, J = 7.9Hz, 2H), 5.97 (br d, J = 8.2 Hz, 1H), 5.69 (br d, J = 7.0 Hz, 2H), 5.59(s, 2H), 4.35-4.26 (m, 1H), 3.79 (br d, J = 11.6 Hz, 2H), 3.69 (s, 1H),3.28 (br t, J = 6.3 Hz, 2H), 3.25- 3.16 (m, 2H), 2.67-2.58 (m, 1H),1.79-1.70 (m, 2H), 1.67-1.52 (m, 2H), 1.44-1.35 (m, 2H), 1.31-1.20 (m,2H), 1.08-0.96 (m, 2H), 0.75 (t, J = 7.3 Hz, 3H) 877 14 190 440.23 1.0 δ7.83 (s, 1H), 7.49 (br d, J = 8.2 Hz, 1H), 7.39 (br d, J = 8.2 Hz, 2H),7.01 (br d, J = 7.9 Hz, 2H), 5.89 (br s, 2H), 4.52-4.39 (m, 1H), 4.28(s, 2H), 4.22-4.18 (m, 2H), 4.18-4.14 (m, 1H), 3.86 (br d, J = 7.6 Hz,1H), 3.33-3.26 (m, 1H), 3.26-3.21 (m, 1H), 3.20 (s, 3H), 1.70-1.63 (m,2H), 1.50-1.40 (m, 2H), 1.05-0.94 (m, 2H), 0.74 (t, J = 7.3 Hz, 3H) 87814 1107 426.19 1.07 δ 7.60-7.56 (m, 1H), 7.16 (br d, J = 7.9 Hz, 2H),6.91 (br d, J = 8.2 Hz, 2H), 5.92 (br d, J = 8.5 Hz, 1H), 5.75-5.63 (m,2H), 5.59 (s, 2H), 4.35- 4.26 (m, 1H), 4.17-4.10 (m, 1H), 3.33-3.27 (m,1H), 3.16 (s, 1H), 2.98 (s, 1H), 2.70 (br t, J = 6.1 Hz, 2H), 1.67-1.53(m, 2H), 1.42-1.33 (m, 2H), 1.02-0.94 (m, 2H), 0.73 (t, J = 7.3 Hz, 3H)879 14 370 483.25 1.13 δ 7.92-7.87 (m, 1H), 7.86 (s, 1H), 7.55 (br d, J= 8.3 Hz, 1H), 7.34-7.28 (m, 3H), 7.01 (d, J = 8.0 Hz, 2H), 5.91 (br s,2H), 4.54-4.44 (m, 1H), 3.72-3.70 (m, 2H), 3.64 (br s, 2H), 3.34 (dt, J= 10.9, 5.6 Hz, 2H), 3.30-3.24 (m, 2H), 3.12 (br s, 2H), 1.71 (q, J =6.1 Hz, 2H), 1.54- 1.44 (m, 2H), 1.09-0.96 (m, 2H), 0.77 (t, J = 7.3 Hz,3H)

The foregoing detailed description of the invention includes passagesthat are chiefly or exclusively concerned with particular parts oraspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious figures and descriptions herein relate to specific embodimentsof the invention, it is to be understood that where a specific featureis disclosed in the context of a particular figure or embodiment, suchfeature can also be used, to the extent appropriate, in the context ofanother figure or embodiment, in combination with another feature, or inthe invention in general.

Further, while the present invention has been particularly described interms of certain preferred embodiments, the invention is not limited tosuch preferred embodiments. Rather, the scope of the invention isdefined by the appended claims.

ACRONYMS AND ABBREVIATIONS

Table C provides a list of acronyms and abbreviations used in thisspecification, along with their meanings.

TABLE C ACRONYM OR ABBREVIATION MEANING OR DEFINITION AIBNAzobisisobutyronitrile Aq. Aqueous Boc t-Butyloxycarbonyl BOP(Benzotriazol-1-yloxy)tris (dimethylamino)- phosphoniumhexafluorophosphate (V) DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCMDichloromethane DIAD Diisopropyl azodicarboxylate DIPEA, DIEAN,N-diisopropylethylamine, also known as Hünig's base DMFN,N-dimethylformamide DMSO Dimethyl sulfoxide FmocFluorenylmethyloxycarbonyl HATU Hexafluorophosphate AzabenzotriazoleTetramethyl Uronium; 1-[Bis (dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate Hunig's base See DIPEA, DIEA HPLC High pressureliquid chromatography LCMS, LC-MS, Liquid chromatography-massspectrometry LC/MS MS Mass spectrometry MsCl Methanesylfonyl chloride,mesyl chloride NBS N-Bromosuccinimide NMR Nuclear magnetic resonance PEGPoly (ethylene glycol) PTFE Poly (tetraflurorethylene) RT (in thecontext of Room (ambient) temperature, circa 25° C. reaction conditions)RT (in context of Retention time, in min liquid chromatography) Sat.Saturated Soln Solution TBDPS tert-Butyldiphenylsilyl TEAATriethylammonium acetate TFA Trifluoroacetic acid THF Tetrahydrofuran

REFERENCES

Full citations for the following references cited in abbreviated fashionby first author (or inventor) and date earlier in this specification areprovided below. Each of these references is incorporated herein byreference for all purposes.

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What is claimed is:
 1. A compound having a structure according toformula I

wherein each X¹ is independently N or CR²; X² is O, CH₂, NH, S, orN(C₁-C₃ alkyl); R¹ is H, CH₃(CH₂)₁₋₃, CH₃(CH₂)₀₋₁O(CH₂)₂₋₃,CH₃(CH₂)₀₋₃C(═O), CH₃(CH₂)₀₋₁O(CH₂)₂₋₃C(═O),

R² is H, O(C₁-C₃ alkyl), C₁-C₃ alkyl, Cl, F, or CN; R³ is H, halo, OH,CN, NH₂, NH(C₁-C₅ alkyl), N(C₁-C₅ alkyl)₂, NH(CH₂)₀₋₁(C₃-C₆ cycloalkyl),NH(C₄-C₈ bicycloalkyl), NH(C₆-C₁₀ spirocycloalkyl), N(C₃-C₆cycloalkyl)₂, NH(CH₂)₁₋₃(aryl), N((CH₂)₁₋₃(aryl))₂, a cyclic aminemoiety having the structure

 a 6-membered aromatic or heteroaromatic moiety or a 5-memberedheteroaromatic moiety; wherein an alkyl, cycloalkyl, bicycloalkyl,spirocycloalkyl, cyclic amine, 6-membered aromatic or heteroaromatic, or5-membered heteroaromatic moiety is optionally substituted with one ormore substituents selected from OH, halo, CN, (C₁-C₃ alkyl), O(C₁-C₃alkyl), C(═O)(Me), SO₂(C₁-C₃ alkyl), C(═O)(Et), NH₂, NH(Me), N(Me)₂,NH(Et), N(Et)₂, and N(C₁-C₃ alkyl), (CH₂)₁₋₂OH, (CH₂)₁₋₂OMe; and acycloalkyl, bicycloalkyl, spirocycloalkyl, or cyclic amine moiety mayhave a CH₂ group replaced by O, S, NH, N(C₁-C₃ alkyl), or N(Boc); m is 0or 1; and n is 1, 2, or 3; or a pharmaceutically acceptable saltthereof.
 2. A compound according to claim 1, wherein the group R¹ isselected from the group consisting of


3. A compound according to claim 1, wherein the group R¹ is


4. A compound according to claim 1, wherein the moiety

is selected from the group consisting of


5. A compound according to claim 1, wherein the moiety


6. A compound according to claim 1, wherein the group R³ is selectedfrom the group consisting of Cl, H,


7. A compound according to claim 1, wherein R³ is a moiety

selected from the group consisting of


8. A compound according to claim 1, having a structure according toformula (I′)


9. A compound according to claim 1, having a structure according toformula Ia


10. A compound according to claim 9, wherein R¹ is


11. A compound according to claim 1, having a structure according toformula Ib


12. A compound according to claim 11, wherein


13. A compound according to claim 1, which is covalently bonded to apoly(ethylene glycol) moiety between 2 kDa and 40 kDa in size.
 14. Amethod of treating a cancer, comprising administering to a patientsuffering from such cancer a therapeutically effective combination of ananti-cancer immunotherapy agent and a compound according to claim
 1. 15.A method according to claim 16, wherein the anti-cancer immunotherapyagent is an antagonistic anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody.16. A method according to claim 15, wherein the cancer is lung cancer(including non-small cell lung cancer), pancreatic cancer, kidneycancer, head and neck cancer, lymphoma (including Hodgkin's lymphoma),skin cancer (including melanoma and Merkel skin cancer), urothelialcancer (including bladder cancer), gastric cancer, hepatocellularcancer, or colorectal cancer.
 17. A method according to claim 15,wherein the anti-cancer immunotherapy agent is ipilimumab, nivolumab, orpembrolizumab.